Cloning in

Eukaryotic
cell
Dr. Hanaa sh
erief
 Gene Cloning in Eukaryotes
Most of studies on gene cloning have been carried out in
bacteria. In addition, many difficulties are associated with them,
for example (i) correction of introns of eukaryotic mRNAs, (ii)
failure of transfer of equal number of plasmids into daughter
cells during cell division and yield of two types of cells, one with
plasmid and the second without plasmid, post-translational
modification (e.g. inhibition of proteolysis, addition of
oligosaccharides to specific sites on the polypeptide chain, the
glycosylation), (iii) expression to ensure for the presence of a
stable plasmid in the bacterium when applied on large scale for
commercial applications.
0 With these prospects the attraction of the use of eukaryotic
cells is obvious. In recent years, considerable efforts have
been made for the improvement of crop plants through
genetic engineering. Many works on various aspects of
building up of vectors and expression of insert genes in
eukaryotic cells are in progress.
 Recombinant DNA technology
in eukaryotes
0 The techniques for gene manipulation, cloning, and
expression were first developed in bacteria but are now
applied routinely in a variety of model eukaryotes. The
genomes of eukaryotes are larger and more complex than
those of bacteria, so modifications of the techniques are
needed to handle the larger amounts of DNA and the array
of different cells and life cycles of eukaryotes. For example,
some eukaryotic proteins cannot be easily expressed in
large amounts in bacteria, and eukaryotic expression
systems need to be employed.
❖ The recent exciting advances in recombinant DNA
cloning technology in eukaryotic cell encouraged us
to undertake the development of methods for the
conversion of polyadenylylated messenger RNA into
double-stranded DNA which could then be inserted
into bacterial plasmids and cloned.
This would provide large quantities of pure structural
gene sequences in the form of duplex DNA,
permitting the facile application of new rapid
sequence techniques employing restriction
endonucleases.
 mRNA extraction
❑ Firstly, the mRNA is obtained and purified from the
rest of the RNAs. Several methods exist for
purifying RNA such as trizol extraction
and column purification.
❑ Column purification is done by using oligomeric
dT nucleotide coated resins where only the mRNA
having the poly-A tail will bind.
❑ The rest of the RNAs are eluted out. The mRNA is
eluted by using eluting buffer and some heat to
separate the mRNA strands from oligo-dT.
 cDNA construction
❑ Once mRNA is purified, oligo-dT (a short sequence of deoxy-thymine
nucleotides) is tagged as a complementary primer which binds to the
poly-A tail providing a free 3'-OH end that can be extended by reverse
transcriptase to create the complementary DNA strand.
❑ Now, the mRNA is removed by using a RNAse enzyme leaving a single
stranded cDNA (sscDNA).
❑ This sscDNA is converted into a double stranded DNA with the help
of DNA polymerase. However, for DNA polymerase to synthesize a
complementary strand a free 3'-OH end is bacterial plasmids.
❑ cDNA is produced from fully transcribed mRNA found in the nucleus and
therefore contains only the expressed genes of an organism. Similarly,
tissue specific cDNA libraries can be produced.
❑ In eukaryotic cells the mature mRNA is already spliced, hence the cDNA
produced lacks introns and can be readily expressed in a bacterial cell.
While information in cDNA libraries is a powerful and useful tool since
gene products are easily identified, the libraries lack information
about enhancers introns, and other regulatory elements found in
a genomic DNA library.
 Construction of cDNA Libraries
A genomic DNA library is a collection of cloned DNA fragments
representing all of the DNA of an organism.
A cDNA library (complementary DNA), is a collection of cloned DNA
fragments corresponding to all mRNAs transcribed in a certain tissue
or organism. Libraries can be constructed using plasmid cloning vectors.

. In Step 1, mRNA isolated by

oligo-dT affinity chromatography
is hybridized via its 3' poly(A) tail
to an oligo-dT primer.
In Step 2, RT synthesizes the
first cDNA strand.
In Step 3, RNA is destroyed and
a poly(dG) tail is added by
terminal transferase.

In Step 4, the cDNA is

hybridized to an oligo-dC primer.
 Construction of cDNA Libraries
In Step 5, a DNA
polymerase is used to
synthesize the second
strand of the cDNA.
In Step 6, EcoRI sites
that might be present
within the mRNA coding
region are protected by
methylation using EcoRI
methylase.
In Step 7, unmethylated
EcoRI linkers, that
encode EcoRI restriction
sites, are ligated to the
ends of the fragment.
In Step 8a, the cDNA is
cleaved with EcoRI
restriction enzyme,
generating sticky-ended
cDNA fragments.
 Construction of cDNA Libraries
In the last steps of cDNA library construction,
In (Step 8b) the plasmid vector is cut with EcoRI restriction enzyme,
and then the EcoRI-cut cDNA and plasmid are ligated together (Step
9). Finally, the E. coli host strain is transformed and cells are plated
(Step 10) on selective medium.
To be complete, both genomic and cDNA libraries for higher
eukaryotes must contain on the order of a million individual clones.
 Screening cDNA Libraries
To screen a plasmid library colonies
representing each cloned DNA
first are plated on a number of petri
plates. Library DNA then is lifted
onto nitrocellulose membranes which
serve as replicas of the plates. Bound
DNA is denatured and hybridized with
a radioactively-labeled single-strand
DNA probe .
After washing, spots corresponding
to colonies containing the DNA of
interest are detected by
autoradiography. Because not all DNA
gets lifted onto the membranes, DNA
for the clone can be purified from the
residual colony on the original plate.
Note, that oligonucleotide probes
must only be ~ 20 nucleotides long to
recognize unique sequences even in
genomic DNA.
The probe sequence can be derived
from genome sequencing databases, or
designed based on the known
sequence of a protein.
1-Purification of mRNA.

2- Synthesis of cDNA.

3-Synthesis of
Double-Stranded Globin DNA.

4-Cleavage of Hairpin
Structures.

5-Attachment of
Homopolymers to
Double-Stranded Globin
and Plasmid DNA.

The isolation and insertion of rabbit globin gene sequences into Escherichia coli
plasmid DNA and cloned the chimeras.
 Simple subcloning
❑ The antibiotic resistance gene and an origin of replication are
found in many plasmids, but our actual example contains a chimeric
gene as well, our gene of interest. It has already been assembled
from the three basic parts, a promoter, a coding region and a 3’end
carrying a polyadenylation signal. This tells us that the gene is
designed to work in a eukaryotic cell (yeast, fungi, protists, plants,
animals).

❑ The promoter is from a virus gene that codes for the 35S coat
protein of cauliflower mosaic virus. The coding region is from a gene
that encodes green fluorescent protein from a jellyfish.

❑ The promoter is designed to work in cauliflower, which is infected by

the virus, another example of an entity that can genetically engineer
plant cells to manufacture more copies of itself. However, the
promoter will also work in other plants, and because it is a very
strong promoter, it is used to express certain genes. The coding
region encodes the green fluorescent protein (GFP), a useful tool in
❑ The polyadenylation signal is essentially a piece of DNA that contains
signals for the processing of the transcript to generate the final messenger
RNA that can be transported out of the nucleus. These three building blocks
build a eukaryotic gene, and because in this case they have been fused
together from different sources, it is a chimeric gene. The polyadenylation
signal comes from a plant gene.
 Transgenic eukaryotes
❑ DNA is introduced into a eukaryotic cell by a variety
of techniques, such as transformation, injection, viral
infection, or bombardment with DNA-coated
tungsten particles .

❑ When exogenously added DNA that is originally from

that organism inserts into the genome, it can either
replace the resident gene or insert ectopically.
 Some of the different ways of
introducing foreign DNA into a
cell.
0 The possibility of transgenic modification of eukaryotes
such as plants and animals (including humans) opens up
many new approaches to research because genotypes can
be genetically engineered to make them suitable for some
specific experiment.
0 Transgenesis, the design of a specific genotype by the
addition of exogenous DNA to the genome, has increased
the scope of breeding in basic genetic research and in
commercial applications.
0 We now turn to some of the specialized recombinant
DNA techniques used in the eukaryotes baker’s yeast,
plants, and animals and in human gene therapy.
 Gene transfer into baker’s yeast
0 The yeast Saccharomyces cerevisiae has become the most
sophisticated eukaryotic model for recombinant DNA technology.

0 One of the main reasons is that the transmission genetics of yeast

is extremely well understood, and the stockpile of thousands of
mutants affecting hundreds of different phenotypes is a valuable
resource when using yeast as a molecular system. In yeast,
another important advantage is the availability of a circular
6.3-kb natural yeast plasmid.

0 transmission genetics (The study of the mechanisms involved in

the passage of a gene from one generation to the next).

0 This plasmid is transmitted to the cellular products

of meiosis and mitosis.
0 Generally bacterial cultures are suitable for the production
of polypeptides on a large scale, but not plant or animal
cells, because of their slow growth rate. But certain
limitations with bacteria have forced to develop its
alternative and, to some extent, it has been overcome with
the use of yeast {Saccharomyces cerevisiae). Yeast is an
unicellular eukaryotic fungus containing a small well
characterized genome. Unlike plant or animal cells, it has
rather fast growth rate and itself is a non-pathogenic
fungus. Most of its gene contain introns which are spliced
during purification of mature mRNA. appears that introns
found in yeast contain sequences for correct splicing as they
are totally absent in higher eukaryotes. Moreover, yeasts can
carry out post-translational modifications such as (removal
of a signal sequence from a precursor polypeptide after the
secretion of cells. This reveals a major advantage of yeasts
over the bacteria.
 Why Saccharomyces cerevisiae?

1) easy to grow and manipulate (like E.coli)

2) biochemistry and cell biology similar between
yeast and “higher” eukaryotes
-- many gene homologs between yeast and humans,
eg. Cell cycle (cancer) genes
3) excellent genetic tools are available in yeast
0 Success in DNA cloning in yeast depends on uptake of foreign
DNA by its spheroplast (naked protoplast when cell wall is
enzymatically removed) in the presence of Ca++ and
polyethyleneglycol (PEG). The spheroplasts develop cell wall
after the DNA is taken up (Hinnen et. al., 1978).
0 Yeast cells contain their own plasmid (Begg, 1978) known as
'2mm circle plasmid' which is used as vector for foreign genes.
It is present in many stains in 50-100 copies per cell.
0 The foreign DNA taken up by yeast is integrated by a specific
crossing over to yeast DNA containing a homologous
sequence on chromosome recognized by the yeast as the
origin of replication (ori). /In some cases segments of yeast
DNA linked with foreign DNA have been demonstrated to
transform very efficiently (about 104 cells) (Hsiao and
Carbon 1979).
0 The transforming molecules replicate autonomously within
the cell DNA sequences of such property are known as Ars
(autonomously replicating sequence) fragment. Ars
fragments have also been isolated from a number of
eukaryotic organisms such as Caenhaorabditis elegans,
Dictyostelium discoideum, Drosophila melanogaster, Zea
mays, etc (Stinchcomb et al, 1980).
0 However, plasmids have been constructed by using ars
fragments when joined at centromeric sequences (cen region
of about 6-10 Kb DNA) to ensure the replication and stability
in yeast.
0 Cen is needed for DNA molecule to segregate correctly during
meiosis and mitosis. Sometimes the minichromosomes are
lost during meiosis when they are attached by chance to
spindle apparatus. Thus, cen regions of yeast plasmids are
useful fragments because they confer stability (Glover, 1984).
 Shuttle Vector
A shuttle vector is constructed by using bacterial origin of
replication in a yeast plasmid which can be manipulated and
cloned in bacteria. Finally they are transferred into yeast cells
for the possible expression of eukaryotic genes . The gene for
B-lactamase is an example of a prokaryotic gene that is
expressed in yeast.
 Kado and Tait (1983) have described the properties of gene
shuttle vector as below :
(i) The vector must replicate in many organism (e.g. bacteria, yeast and plants) to
facilitate the isolation and characterization of genes;

(ii) The vector must be easily recognized by selectable markers;

(iii) The vector should be small in size to accommodate DNA inserts;

(iv) Cloned genes should be easily detected;

(v) Useful quantities of vector must be easily obtained;

(vi) The vector must be stable, non-pathogenic and non-stress-inducing;

(vii) The vector must effectively deliver genetic information for stable maintenance in
alternate derived reciptients;

(viii) The introduced genetic information should be stably maintained as a new heritable
determinant.
Fig. Map of shuttle vector
constructed by yeast and E.coli. Ars.
autonomously replicating sequence;
Cen, centromere of yeast; leu 2,
complements of a defective gene
encoding for leucin; on. origin of
replication in prokaryote; Ampr,
amphcillin resistance
 Yeast transformation

❑ Electroporation, or chemical competence (Lithium

chloride/PEG treatment).
❑ Isolate transformants using nutritional markers:
0 His3, Leu2, Trp1--amino acid biosynthetic genes
0 Ura3--nucleotide biosynthetic gene.
(these require auxotrophic yeast strains)
❑ Auxotrophic :the inability of an organism to synthesize a
particular organic compound required for its growth. An auxotroph
is an organism that displays this characteristic; auxotrophic is the
corresponding adjective. Auxotrophy is the opposite of
prototrophy, which is characterized by the ability to synthesize all
the compounds needed for growth.
❑ antibiotic resistance markers
Simplified representations of four different kinds of plasmids used in yeast. Each is
shown acting as a vector for some genetic region of interest, which has been inserted
into the vector. The function of such segments can be studied by transforming a yeast
strain of suitable genotype. Selectable markers are needed for the routine detection
of the plasmid in bacteria or yeast. Origins of replication are sites needed for the
bacterial or yeast replication enzymes to initiate the replication process. (DNA
derived from the 2-μm natural yeast plasmid has its own origins of replication.)
Yeast integrative plasmids(YIP)
1) Propagate and engineer using E. coli as a host

2) No yeast origin of replication (MUST integrate)

3) Genome engineering through homologous

recombination.
❑ The simplest yeast vectors are derivatives of bacterial plasmids
into which the yeast locus of interest has been inserted . When
transformed into yeast cells, these plasmids insert into yeast
chromosomes generally by homologous recombination with
the resident gene and by either a single or a double crossover.
❑ As a result, either the entire plasmid is inserted or the
targeted allele is replaced by the allele on the plasmid. Such
integrations can be detected by plating cells on a medium that
selects for the allele on the plasmid.
❑ Because bacterial plasmids do not replicate in yeast,
integration is the only way to generate a stable
modified genotype with the use of these vectors.
❑ Yeast vectors can be integrative, can autonomously replicate, or
can resemble artificial chromosomes, allowing genes to be
isolated, manipulated, and reinserted in molecular genetic
analysis.
Two ways in which a recipient yeast strain bearing a defective X− can be
transformed by a plasmid bearing an active allele (gene X+). The mutant
site of gene X− is represented as a vertical dark green bar.
 Yeast integrative plasmid:
homologous recombination
0 No yeast replicon, can transform but
cannot replicate.
0 Requires integration into chromosome for
propagation, but very stable

0 Useful for manipulating (eg. deleting)

genes on the chromosome
 The first demonstration of a yeast
integrative plasmid: leu2
complementation
Wild type yeast: grows on minimal medium lacking
leucine because it has the leucine biosynthetic genes

Leu2 yeast: a mutation in the leu2 gene, it knocks out

leucine biosynthesis, therefore no growth without
leucine

pYeLeu10: a plasmid (with no yeast replicon) that

contains the yeast Leu2 gene--can it complement the
Leu2 mutant yeast????
 The experiment:
•Transform Leu2 mutant cells, using pYeLeu10 (which
contains an intact Leu2 gene)
•Selection for growth in the absence of leucine (leu
dropout plates)
•What will grow? Only those cells that can replicate the
Leu2 gene coming from the plasmid

Results: some transformants survive….

Three ways for the leu2 gene to be
maintained
(all via integration)

Mutant Leu2

Double (1
crossover

Single (2
crossover
(kinds 3) (integration)

Random (3
insertion
 Yeast Episomal plasmid (YEP)
❑ Contains naturally occuring “2 micron circle” origin of replication
❑ High copy number: 50-100/cell
❑ Shuttle vector -- replicon for E. coli.
❑ 2-μm plasmid is used as the basic vector and other bacterial and yeast
segments are spliced into it , then a construct having several useful
properties is obtained. First, the 2-μm segment confers the ability to
replicate autonomously in the yeast cell, and insertion is not necessary
for a stable transformation. Second, genes can be introduced into yeast,
and their effects can be studied in that organism; then the plasmid can
be recovered and put back into E. coli, provided that a bacterial
replication origin and a selectable bacterial marker are on the plasmid.
❑ Such shuttle vectors are very useful in the routine cloning and manipulation
of yeast genes.
❑ A shuttle vector is a vector (usually a plasmid) constructed so that it can
propagate in two different host species.
❑ The main advantage of these vectors is they can be manipulated in E. coli,
then used in a system which is more difficult or slower to use (e.g. yeast).
 A yeast episomal plasmid

Shuttle vector: has sequences

allowing replication in E.coli
 Yeast Centromeric plasmid
(Ycp)
0 Contains yeast ars (autonomously
replicating sequence) for replication
0 Contains yeast centromere for proper
segregation to daughter cells (Two identical
cells formed by asexual division of a cell).

0 Low copy number, ~1 per cell (good for

cloning genes that are toxic or otherwise
affect cell physiology)
0 Stable, shows mendelian segregation.
0 Centromeric plasmids can be used to study the
regulatory elements upstream of a gene
 YAC: yeast artificial
chromosome
0 Replicates as chromosome:
has centromere and
telomeres
0 Useful for cloning very large
pieces of DNA
0 . Large DNA (>100 kb) is ligated
between two arms Each arm
ends with a yeast telomere so
that the product can be
stabilized in the yeast cell.
Interestingly, larger YACs are
more stable than shorter ones,
which favors cloning of large
stretches of DNA.
0 2. One arm contains an
autonomous replication
sequence (ARS), a centromere
(CEN) and a selectable marker
(trp1). The other arm contains a
second selectable marker (ura3).
 Transformation in Filamentous
0 Fungi
Transformation through plasmid of E. coli was first
described in protoplasts of S. cerevisiae (Hinnen et al., 1978)
and then in N. crassa. However, the situation in filmentous
fungi is different from the others as there is no convincing
evidence for autonomously replicating plasmids. But
sequence having functions similar to ARS have been
isolated.
0 Method of transformation is similar as for plant cells.
Following are the examples where transformations in
protoplasts have been carried out: A. nidulans, A. niger, A.
oryzae, Cephalosporium acremonium, Coprinus cinereus,
Glomerella cingulata, Gaeumannomyce graminis, Mucor sp.,
Penicillium chrysogenum, Septoriu nodorum, etc.
 Application of Transformation of Fungal
Protoplasts
0 Mainly the transformation approaches are used (i) for the
analysis of promoters and construction of expression vectors,
and (ii) Biotechnological application in the expression of
foreign genes in fungi. Analysis of promoter involves the
construction of vector which have different pieces of
promoters. These can be cut by restriction enzymes and joined
with DNA ligase. By this method changes in promoter region
are also possible and this can be achieved through mutagenesis.
Thus, a strain can be improved resulting in overproduction of
the products (Peberdy, 1989).
0 Adopting these approaches many mammalian genes have now
been expressed in S. cerevisiae and aspergilli through
expression vector (i.e. vector which include sequences
promoting high level of transcription of the gene and secretion
of the gene products).
0 Several companies in the U.S.A. and Europe have demonstrated
the expression of foreign DNA introduced in Aspergillus sp.
through transformation.
0 Upshall et al. (1987) have constructed a plasmid pMl59 for the
secretion of human tissue plasminogeil activater by A. nidulans.
Yield of this protein was about 100mg/liter culture filtrate.
Similarly, Gwynne et al. (1987) found the secretion of human
interferon by transformed A. nidulans mycelia to about 1
mg/liter.
 Gene transfer in Plants
0 The Ti plasmid :The only vectors routinely used to produce
transgenic plants are derived from a soil bacterium called
Agrobacterium tumefaciens. This bacterium causes what is
known as crown gall disease, in which the infected plant
produces uncontrolled growths (tumors, or galls), normally
at the base (crown) of the plant.
0 The key to tumor production is a large (200-kb) circular
DNA plasmid—the Ti (tumor-inducing) plasmid. When the
bacterium infects a plant cell, a part of the Ti plasmid—a
region called T-DNA—is transferred and inserted,
apparently more or less at random, into the genome of the
host plant .
In the process of causing crown gall disease, the bacterium Agrobacterium
tumefaciens inserts a part of its Ti plasmid—a region called T-DNA—into a
chromosome of the host plant.
❑ The functions required for this transfer are outside the
T-DNA on the Ti plasmid. The T-DNA itself carries several
interesting functions, including the production of the tumor
and the synthesis of compounds called opines.

❑ Opines are actually synthesized by the host plant under the

direction of the T-DNA. The bacterium then uses the opines
for its own purposes, calling on opine-utilizing genes on the
Ti plasmid. Two important opines are nopaline and
octopine; two separate Ti plasmids produce them.
Simplified representation of the major regions of the Ti plasmid of A. tumefaciens.
The T-DNA, when inserted into the chromosomal DNA of the host plant, directs the
synthesis of nopaline, which is then utilized by the bacterium for its own purposes.
T-DNA also directs the plant cell to divide in an uncontrolled manner, producing a
tumor.
0 The natural behavior of the Ti plasmid makes it well-suited for
the role of a plant vector. If the DNA of interest could be spliced
into the T-DNA, then the whole package would be inserted in a
stable state into a plant chromosome. This system has indeed
been made to work essentially in this way, but with some
necessary modifications.
0 Ti plasmids are too large to be easily manipulated and cannot
be readily made smaller, because they contain few unique
restriction sites. Consequently, a smaller, intermediate vector
initially receives the insert of interest and the various other
genes and segments necessary for recombination, replication,
and antibiotic resistance.
0 It retains the left-hand border (L) of its T-DNA, which will be
used as the crossover site for incorporation of the intermediate
vector.
0 The intermediate vector has had a convenient cloning segment
spliced in, containing a variety of unique restriction sites. Also
spliced into the vector are a selectable bacterial gene (spcR) for
spectinomycin resistance; a bacterial kanamycin-resistance gene
(kanR), engineered for expression in plants; and two segments of
T-DNA.

0 The second T-DNA segment comes from near the left-hand

border and provides a section for recombination with a
homologous part of region L, which was retained in the disarmed
Ti plasmid.
0 After the intermediate vectors have been introduced into
Agrobacterium cells containing the disarmed Ti plasmids (by
conjugation with E. coli), plasmid recombinants (cointegrates)
can be selected by plating on spectinomycin. The selected
bacterial colonies will contain only the Ti plasmid, because the
intermediate vector is incapable of replication in Agrobacterium.
To produce transgenic plants, an intermediate vector of manageable size is used to
clone the segment of interest. In the method shown here, the intermediate vector is
then recombined with an attenuated (“disarmed”) Ti plasmid to generate a
cointegrate structure bearing the insert of interest and a selectable plant
kanamycin-resistance marker between the T-DNA borders, which is all the T-DNA that
❑ after spectinomycin selection for the cointegrates, bacteria
containing the recombinant double, or cointegrant, plasmid
are then used to infect cut segments of plant tissue, such as
punched-out leaf disks. If bacterial infection of plant cells takes
place, any genetic material between the left and right T-DNA
border sequences can be inserted into the plant chromosomes.

❑ If the leaf disks are placed on a medium containing

kanamycin, the only plant cells that will undergo cell division
are those that have acquired the kanR gene from T-DNA
transfer. The growth of such cells results in a clump, or callus,
which is an indication that transformation has taken place.
These calli can be induced to form shoots and roots, at which
time they are transferred to soil where they develop into
transgenic plants .
The generation of a transgenic plant through the growth of a cell
transformed by T-DNA.
 Gene transfer in dicots by
using T-DNA as vector.
0 It is not possible to transfer Ti-plasmid into plant cell because
of its large size, most probably upto 235 Kb.
0 Therefore, T-DNA is excised and used as foreign DNA for
expression in plant cells. From bacteriological point of view
opine production and development of gall are not required for
the successful integration and expression of DNA. Hence,
replacement of nos and ops genes with a foreign DNA fragment
retaining the opine synthase promoter for expression of
inserted DNA fragment has no harm.
 Gene transfer in dicots by
using T-DNA as vector.
0 Schell and Van Montague (1983) undertook some experiments
where genes for resistance to kanamycin and methatrexate
expressed in cultured callus cells. Generally it is easy to use
opine promoters for the expression of foreign DNA. It has
become possible that the inserted genes, like other plant genes,
will express only at certain stages or specific tissues of plants
when they are with specific regulated promoters.
0 There are reports of replacement of opine synthase promoters
by regulatory promoters obtained from plants. Murai et al.
(1983) have introduced gene for bean plant storage protein, the
phaseolin (which is supplemented with methionine codon), into
the cells of sunflower plant after transformation with
Ti-plasmid where phaseolin gene was under the control of its
own promoters, the octopine synthase.
 Gene transfer in monocots.
0 The limitation of Ti-plasmid is that it is specific only to the
dicot plants. A. tumifaciens does not induce tumour in
monocot plants, however, most of the major crops including
cereals are monocots. Hooykaas Van et al. (1984) discovered
that A. tumifaciens could transfer T-DNA into certain monocots
with the consequences of expression of opine gene within the
plant cell without inducing the tumour. This discovery made it
possible that T-DNA can be expressed into cells of monocot
plants.
Plant cell transformation by ultrasonication

Ultrasonication is done for various biological experiment.

But in some plants this technique has been done for
successful gene delivery.
The Biotechnology Research Center, Beijing (China) has
used this technique for gene transfer in plants like wheat,
tobacco and sugarbeet.
When the cultured explants were sonicated with plasmid
DNA carrying market genes such as cat, nptll, and gus, and
sonicated calli transferred to selective medium, shoots
were grown successfully.
The calli in control set of experiment which were not
sonicated with plasmid did not grow on selective medium
but they died. Transgenic tobacco plants were obtained at a
frequency of 22 per cent.
Liposome-mediated gene transfer
Liposomes are microscopically small sized particles. They
contain a phospholipid bilayers which are concentric in
nature. Liposomes enclose aqeuous chamber and can entrap
water soluble molecules. Therefore, they are called lipid bags.
Many plasmids are enclosed in them. By using polyethylene
glycol (PEG) they may be stimulated to fuse with protoplast. In
several plants like carrot, tobacco, petunia, etc.
This technique has been used for successful transfer of genes.
Due to endocyclosis of liposomes, DNA enters the protoplast.
It gets adhered first to protoplast surface and get fused with
protoplast at the site of surface. Consequently plasmids are
released inside the cell.
There are several advantages in using this technique such as
(i) low toxicity, (ii) protection of DNA/RNA from nucleases that
lyse them, (iii) long stable storage of DNA fragments in
liposome, (iv) applicability in various cell types, and (v) high
level of reproductivity.
Human Gene
Transfer
lec2

Dr. Hanaa Sherief

 The first attempt at modifying human DNA was
performed in 1980 by Martin Cline, but the first
successful nuclear gene transfer in humans,
approved by the National Institutes of Health, was
performed in May 1989.
 The first therapeutic use of gene transfer as well as
the first direct insertion of human DNA into the
nuclear genome was performed by French
Anderson in a trial starting in September 1990.
 Between 1989 and February 2016, over 2,300
clinical trials had been conducted, more than
half of them in phase I
 Not all medical procedures that introduce
alterations to a patient's genetic makeup can
be considered gene therapy.
 Bone marrow transplantation and organ
transplants in general have been found to
introduce foreign DNA into patients.
 Gene therapy is defined by the precision of
the procedure and the intention of direct
therapeutic effects.
Human gene transfer (HGT)
 Human gene transfer (HGT) is defined as
the transfer of genetic material (DNA or
RNA) into a person.
 This experimental technique is being
studied to see whether it could treat
certain health problems by
―compensating for defective genes,
prompting the body to make a potentially
therapeutic substance, or triggering the
immune system to fight disease.
 HGT research is sometimes called "gene therapy"
research, although many researchers and ethicists
prefer not to use the term “therapy” to refer to
products which are still investigational. HGT
research is now being tested in a wide number of
therapeutic indications.
Gene transfer and gene
therapy
 Gene transfer and gene therapy are not
synonyms. Gene transfer is a broad
category encompassing technique of
introducing a genetic sequence with any
function, for example, the transfer of a
fluorescent protein marker into a cell for
diagnostic purposes. The process of gene
transfer may or may not have a
therapeutic purpose or demonstrated
therapeutic effect.
Gene transfer and gene
therapy
 Gene therapy is the clinical application of gene
transfer, with the intent to produce beneficial
health consequences. In research ethics,
 the term therapy is generally reserved for a
product or intervention with demonstrated
safety and efficacy—it is not applied to
interventions that are still being investigated, a
distinction that is important,
 giventhat all gene transfer products are investigational;
none has received a Biologic Licensing Application1
(BLA) approval by the U.S. Food and Drug
Administration (FDA) thus far.

 Conceptually, gene transfer is disarmingly simple.

Introduce a gene into a cell, tissue, or organ, and a
functional protein product will express a protein useful
for clinical diagnostics or nonfunctional pathway to
ameliorate treatment of a disease.
Human gene transfer
 Human gene transfer is the process of
transferring genetic material (DNA or
RNA) into a person. DNA may be
transferred as "naked" DNA, encapsulated
DNA, or DNA within another organism,
such as a virus. Use of retroviral vectors in
humans also constitutes human gene
transfer when the virus contains enzymes
that result in a DNA copy of the RNA
genome.
 All human gene transfer protocols subject to
the NIH Guidelines must be registered with
the NIH and be reviewed and approved by
institutional oversight bodies such as the
Institutional Biosafety Committee IBCs and
Institutional Review Board (IRB).
Different gene delivery systems
Germ line gene therapy
 The technology of this type of gene therapy is simple as
genetic abnormalities can be corrected by direct
manipulation of germline cells with no targeting, and not
only achieve a cure for the individual treated, but some
gametes could also carry the corrected genotype.

 Although it almost never has been tested on humans,

some different transgenic techniques have been used
on other species, which include following
 (1) Gene delivery to the nuclei taken from somatic cells at
metaphase stage.
 (2) Ex vivo alteration of egg cells, following in vitro
fertilization.
 (3)Manipulation of embryonic stem cells of mouse during in
vitro culture by different gene delivery systems.
 (4)Pronuclear microinjection of exogenous DNA solution by a
glass needle.
 (5)Transgenic delivery into sperm cells by direct or indirect
injection to testis .
 Result in permanent changes.
 Potential for offering a permanent therapeutic effect for all
who inherit the target gene.
 Possibility of eliminating some diseases from a particular
family.
Somatic gene therapy

 Somatic gene therapy involves the insertion of

genes into diploid cells of an individual where the
genetic material is not passed on to its progeny.
Somatic cell therapy is viewed as a more
conservative, safer approach because it affects
only the targeted cells in the patient, and is not
passed on to future generations
 Different vector systems for gene
delivery
 GT utilizes the delivery of DNA into cells, which can
be accomplished by a number of methods.
 The two major classes of methods :
 recombinant viruses – VIRAL VECTOR
 naked DNA or DNA complexes – NONVIRAL
VECTOR
Viral vectors

 One of the successful gene therapy systems available today are viral
vectors, such as retrovirus, adenovirus (types 2 and 5), adeno-associated
virus, herpes virus, pox virus, human foamy virus (HFV), and
lentivirus.
 [All viral vector genomes have been modified by deleting some
areas of their genomes so that their replication becomes deranged
and it makes them more safe, but the system has some problems,
such as their marked immunogenicity that causes induction of
inflammatory system leading to degeneration of transducted tissue;
and toxin production, including mortality, the insertional
mutagenesis; and their limitation in transgenic capacity size.
 During the past few years some viral vectors with specific receptors
have been designed that could transfer the transgenes to some other
specific cells, which are not their natural target cells (retargeting).]
Steps of Viral vectors
 Virus bind to their hosts and introduce their genetic material into the
host cell.
 Plausible strategy for gene therapy, by removing the viral DNA and
using the virus as a vehicle to deliver the therapeutic DNA.
 The viruses used are altered to make them safe, although some risks
still exist with gene therapy.
 Many GT clinical trials rely on retroviruses or adenoviruses to
deliver the desired gene.
 Other viruses used as vectors include adeno-associated viruses,
lentiviruses, pox viruses, alphaviruses, and herpes viruses.
 Differ in how well they transfer genes to the cells they recognize
and are able to infect, and whether they alter the cell’s DNA
permanently or temporarily.
VIRAL VECTOR
 Are a tool commonly used by molecular
biologists to deliver genetic material into cells.
 Can be performed in vivo or in vitro.
 Viruses have evolved specialized molecular
mechanisms to efficiently transport their
genomes inside the cells they infect.
 Delivery of genes by a virus is termed
transduction and the infected cells are
described as transduced.
Nonviral delivery systems
 Non viral systems comprise all the physical and chemical
systems except viral systems and generally include either
chemical methods, such as cationic liposomes and
polymers, or physical methods, such as gene gun,
electroporation, particle bombardment, ultrasound
utilization, and magnetofection.
 Efficiency of this system is less than viral systems in gene
transduction, but their cost-effectiveness, availability, and
more importantly less induction of immune system and no
limitation in size of transgenic DNA compared with viral
system have made them more effective for gene delivery
than non-viral delivery systems to date.
Physical methods of nonviral gene
delivery

 Physicalmethods applied for in vitro and

in vivo gene delivery are based on
making transient penetration in cell
membrane by mechanical, electrical,
ultrasonic, hydrodynamic, or laser-based
energy so that DNA entrance into the
targeted cells is facilitated.
 Naked DNA

 Naked DNA alone is able to transfer a gene (2–19

kb) into skin, thymus, cardiac muscle, and
especially skeletal muscle and liver cells when
directly injected, also it has been applied direct
Long-term expression has been observed in
skeletal muscle following injection for more than 19
months. Single injection yields transgenic
expression in less than 1% of total myofibers of the
muscle but multiple injection would improve it.
Although naked DNA injection is a safe and simple
method, its efficiency for gene delivery is low so it
is only proper for some applications, such as DNA
vaccination.
Chemical nonviral delivery systems
 Chemical systems are more common than physical
methods and generally are nanomeric complexes,
which include compaction of negatively charged
nucleic acid by polycationic nanomeric particles,
belonging to cationic liposome/micelle or cationic
polymers
REMAINING CONCERNS IN
GENE TRANSFER RESEARCH

 Although dramatic advances have taken place regarding the techniques of

human gene transfer and mechanisms of action, some gaps in scientific
knowledge remain.
 First, although active viral vectors are designed to be replication
incompetent and no longer pathogenic, predicting severe immune response
remains difficult.
 Second, each component of a transfer carries its own risk, thus complicating
risk assessments. For example, the cases of leukemia that arose in a trial for
X-linked severe combined immunodeficiency (discussed above) may have
been attributable to the combined toxicity of the vector and the transgene
(Baum et al., 2003).
 Third, permanent genetic modification may involve life-long exposure risks.
There are few long-term studies of any low-level toxic effects of a transgene
product or assessments of cumulative effects on the health of a research
participant over time. Many features of human gene transfer, although not
unique, raise concerns and present complex risks for research participants.
 Risk Assessment

 Even with these pivotal advances and dramatic examples of clinical

success, risk assessment remains difficult in gene transfer research
(Deakin et al., 2010). Some of the theoretical risks have been
invalidated, and some genuine risks have exceeded expectations or
were never uncovered by preclinical studies. For example, as discussed
above, the risk of extreme immune reaction and death was not fully
appreciated by preclinical studies in the case of Jesse Gelsinger.

 The risks of gene transfer products (and cellular therapy products) can
be different from those typically associated with other types of
pharmaceuticals, as seen in current draft guidance from FDA (2013).
In addition, the preclinical data generated for cell or gene therapy
products may not always be as informative as for small molecule
pharmaceuticals, particularly because it usually is not feasible to
conduct traditional preclinical pharmacokinetic studies with cell and
gene therapy products.
 Additional considerations regarding
gene therapy include the following:
 a)In vivo gene therapy can inadvertently target transgene
expression to an unintended and clinically unaffected cell or
tissue type, with a potential for toxicity.
 b)Some gene transfer vectors, such as adeno-associated virus,
introduced into non-dividing cells, such as neurons or striated
muscle, present the potential for lifelong persistence of vector
and transgene expression (Lee et al., 2013). This may also
produce a potential for toxicity, particularly if the sustained
function of gene-modified cells alters relationships with
unmodified cells.
 The potential for off-target toxic effects of modified cells must
be considered.
 For example, one study involving the infusion of T cells that were genetically
engineered to target tumors resulted in unexpected off-target cardiac toxicity. The
engineered T cells bound to cardiac muscle tissue, and the resulting toxic effects
on the heart were lethal to the research participant. Available preclinical models
did not demonstrate this risk (Ertl et al., 2011; Morgan et al., 2010).
 Insertional mutagenesis (genotoxicity) was a predicted risk, and although it is not
unique to gene transfer, in some cases it presented more problems than expected
in clinical studies. In a 2008 trial involving 12 patients with X-linked severe
combined immunodeficiency disorder, 4 patients developed vector-induced T-cell
leukemia (Aiuti and Roncarolo, 2009). Immediate treatment with chemotherapy
to all four patients with leukemia sent three into remission, but one died.
Although 11 participants in the trial survived and regained normal immune
function, the trial results were a major setback for the fiel
Dr Hanaa sherief
Dr Hanaa sherief
Scientific Hurdles

 Some scientific hurdles—such as the absence of

efficient delivery systems, difficulty with sustained
expression, insertional mutagenesis and host
immune reactions—remain formidable challenges
to the field (Kay, 2011).
 Some practical limitations associated with even
the most successful gene transfer techniques
remain to be resolved before any gene transfer
procedure can be demonstrated to be a safe and
effective therapy (Grigsby and Leong, 2010).
Many of the major hurdles have to do with
providing efficient gene delivery.
Scientific Hurdles

 First, the vector uptake and distribution must be

tightly controlled so that expression of the vector-
encoded gene remains within the therapeutic
range—if expression is too low, the functional
protein product may not be produced at a high
enough concentration to effectively restore the
intended biochemical pathway, and if expression is
too high, the research subject may experience toxic
effects. Transcription of the new genetic material
must also remain stable so that the transgene is
expressed as long as necessary to treat the disease.
 Second, a substantial proportion of the
population has been exposed to viruses from
which vectors have been derived (or
engineered), especially adenoviral and adeno-
associated viral vectors.
 Third, gene transfer involves the interaction of
many agents. The combined risk factors
associated with the individual components, risks
that may be amplified by their interaction,
complicate risk assessments (Kimmelman, 2005).
 Finally, permanent genetic modification may
expose patients to lifelong risks. Few long-term
studies have been conducted to detect
potential low-level toxic side effects of gene
transfer products or assess cumulative effects on
patient health over time .
Thank you
Gene transfer (transfection)
methods in animals

Dr. Hanaa SHerief

 Gene transfer in Animals
❑ Some of the animals most extensively used as model systems
for DNA manipulation are Caenorhabditis elegans (a
nematode), Drosophila, and mice. Versions of many of the
techniques considered so far can also be applied in these
animal systems.

❑ Fungal, plant, and animal genes can be cloned and

manipulated in bacteria and reintroduced into the eukaryote
cell, where they generally integrate into chromosomal DNA.

❑ There are several ways of producing transgenic animals. An

example is the production of transgenic mammals is by
injecting special plasmid vectors into a fertilized egg .
 Gene transfer (transfection)
methods in animals

🞂 In bacteria and other microbes, or even in higher

plants, the uptake of genes by cells is often described
as 'transformation'.
🞂 In animals, this term is replaced by the term
'transfection' because the term transformation is used
in animals to describe phenotypic alteration in cells.
🞂 Transfection or gene transfer in animals can be carried
out at the cellular level and the transfected cells may be
used for a variety of purposes including the following :
🞂 (i) Production of chemicals and pharmaceutical drugs.
🞂 (ii) Study of structure and function of genes
🞂 (iii) Production of transgenic animals for increased milk
production, increased growth rate of livestock and fish,
etc., large scale production of valuable proteins (to be
extracted from milk, urine and blood ; this is described as
'molecular farming') and for improvement of wool in
sheep.
🞂 The transfection methods may employ the fertilized
eggs/embryos or the cultured cells. The technique
and the purpose of transfection may differ in the
two cases.
🞂 Transfer of genes to fertilized eggs or embryos. The
transfection of fertilized egg may involve transfer of
whole nuclei, whole chromosomes (or fragments) or
DNA segments
(i) For the transfer of whole
nuclei
🞂 The egg cells are treated with cytochalasin-B and
subjected to centrifugation causing enucleation
(nucleus is removed). Incubation of desirable
karyoplasts with these enucleated eggs leads to the
transfer of whole nuclei in presence of polyethylene
glycol (PEG)
 (ii) For transfer of whole
chromosomes
🞂 The chromosomes are first isolated from
metaphase cells by hypotonic lysis and may
be fractionated using density centrifugation
or flow cytophotometry.
🞂 Individual chromosomes or fragments thus
isolated are then incubated with whole cells
(eggs) for incorporation of chromosomes into
nuclei
 (iii) Microinjection of DNA segments
into fertilized eggs
🞂 This technique is the most commonly used
method . Several hundred copies of the DNA
segment are utilized and after stable
integration, the eggs are used for getting
transgenic animals.
🞂 This method is commonly used for the
production of transgenic mice
Procedure of Microinjection :

🞂 This is the direct introduction of the recombinant DNA

into the host cell. This technique has been used
successfully with both plant and animal cells. In this
procedure the cell is held on a glass capillary by gentle
suction.
🞂 The microinjection needle is made by drawing out a
heated glass capillary to a fine point. Using a
micromanipulator (a mechanical device for fine control
of the capillary) the needle has been inserted into the
nucleus of the host cell.
Disadvantage of Microinjection
🞂 One obvious disadvantage is that this technique is
labour-intensive and not suitable for primary cloning
procedures where large numbers of recombinants are
required.
🞂 However, in certain specialised cases it is an
excellent method for targeting DNA delivery once a
suitable recombinant has been identified and
developed to the point where microinjection is
feasible.
Fig. Microinjection of foreign DNA into a fertilized egg for transfer of a
gene leading to the production of transgenic animals.
Fig. Different steps in the production of transgenic mice through
microinjection of eggs.
Transfer of genes to cultured
cells.
🞂 Transfection of cultured cells (usually stem cells; stem cells are
undifferentiated precursor cells) is often attempted for purposes of
gene therapy, etc. Gene transfer to cultured cells can be achieved either
through the use of retroviruses and other viruses as vectors or by direct
delivery of DNA by any of the following methods :
🞂 (i) coprecipitation of DNA with calcium phosphate.
🞂 (ii) use of complexes, of DNA with polycations or lipids.
🞂 (iii) electroporation (a technique, where cell membrane is made more
permeable by electric shocks.
🞂 (iv) use of gold or tungsten particles coated with DNA and accelerated
to a high speed using a particle gun.
🞂 (v) microinjection of DNA using micro-manipulator
🞂 and (vi) use of liposomes or sphaeroplasts, which are vesicles
carrying DNA. In future more efficient methods of gene transfer to
cultured cells in animals will become available.
Fig. Transfection of stem cells and injection of derived stem cells
into blastocyst, leading to the production of transgenic
 Method of Electroporation:
🞂 Electroporation or electro-permeabilization is
the process of applying electrical field to a living
cell for a brief duration of time in order to create
microscopic pores in the plasma membrane
called electro-pores.
🞂 This technique is used for transferring the
recombinant DNA molecule into wide range of
hosts starting from bacteria to plant (plant
protoplasts) and animal cells.
Principle of electroporation:

🞂 The phospholipid molecules of the plasma

membrane are not static. When we apply elec-
tric field to them their kinetic energy
increases resulting in the increase in the
membrane permeability at certain points.
This is exactly where we see the formation of
electro-pores.
🞂 The recombinant DNA can pass through these
transient pores before they close.
Procedure of electroporation :

🞂 In this process cells are mixed with the recom-

binant DNA and the mixture is placed in a small
chamber with electrodes connected to a
specialized power supply. Then a brief electric
impulse is discharged across the electrodes,
which makes pores (holes) in the plasma
membrane.
🞂 These pores remain for some time and are again
resealed themselves. Recombinant DNA enters
the cell which are removed and plated in fresh
selective medium. The process of selection is
then applied to isolate cells carrying recombinant
DNA.
 Method of Liposome Encapsulation
(Lipofection):

🞂 This technique is found very successful in the

transfection of animal host cells and plant
protoplasts.
Principle of Lipofection :
🞂 Liposomes are microscopic vesicles developed
in a laboratory environment. Each liposome is a
spherical ball like structure made up of
phospholipid bilayers with a hollow central
space, allowing liposomes to interact directly
with cells.
🞂 A liposome can fuse with the cell membrane of
the taken host cell and can deliver its content
to it. The recombinant DNA enclosed in the
liposome vesicles penetrates into the
protoplast of the host cell.
Procedure of Lipofection :
🞂 In this technique the recombinant DNA, which is negatively
charged at a near neutral pH because of its phosphodiester
backbone, is mixed with the lipid molecules with positively
charged (cationic) head groups. The lipid molecules form a
bilayer around the recombinant DNA molecules.
🞂 This results in the formation of liposomes which are further
mixed with the host cells. Most eukaryotic cells are
negatively charged at their surface, so the positively charged
liposomes interact with the cells. Cells take up the lipid-
recombinant DNA complexes, and some of the transfected
DNA enters the nucleus.
Biolistic Particle Delivery System:
🞂 A gene gun or a biolistic particle delivery system is a
device which can directly bombard small particles
coated with the recombinant DNA on the nucleus of
the target cell. This technique is often simply referred
to as bio-ballistics or biolistics and has been success-
fully used in the transfection of both plant and animal
cells.
🞂 In this technique the recombinant DNA is coated with
microscopic tungsten particles known as micro-
projectiles, which are then accelerated on a macro-
projectile by firing a gunpowder charge or by using
compressed gas to drive the macro-projectile.
🞂 At one end of the ‘gun’ there is a small aperture that
stops the macro-projectile but allows the micro-
projectiles to pass through. When directed at cells,
these micro-projectiles carry the DNA into the cell
and, in some cases, stable transformation will occur.
 Method of Calcium Phosphate
Co-Precipitation:
🞂 This technique is used for the transfection of plant and
mostly animal cells. The recombinant DNA is mixed with
calcium chloride in a phosphate buffer at neutral pH. This
results in the formation of recombinant DNA-calcium
phosphate complex which appears as a thin precipitate. This
precipitate is then added to the host cell.
🞂 The precipitate is taken up by the cell by the process of
phagocytosis. The recombinant DNA enters the nucleus and
integrates into the host’s genome. The transfection
efficiency can be increased by exposing the host cell to 10-
20% glycerol or Dimethyl sulfoxide (DMSO).
Method of Sonoporation:
🞂 Sonoporation, or cellular sonication, is the
use of sound (typically ultrasonic frequencies)
for the transfer of recombinant DNA into the
target host cell. This process has been
successfully used in a wide range of host cells
starting from bacteria to plant and animal
cells.
🞂 This employs the acoustic waves to increase
the permeability of the plasma membrane.
Taking the advantage of this situation the re-
combinant DNA enters the host cell.
Method of Optical
Transfection
🞂 :
🞂 Optical Transfection is the process of introducing
nucleic acids into cells using light. This has been
successful in transfecting animal cells. In this
technique the plasma membrane of the host cell
is exposed to the highly focused laser beam for a
small amount of time (typically tens of
milliseconds to seconds), generating a transient
pore on the membrane called photo-pore.
🞂 Through the photo-pore the recombinant DNA
can enter the host cell.
Method of Impalefection:

🞂 Impalefection is a method of gene delivery

using Nano materials, such as carbon Nano
fibres, carbon nanotubes, nanowires, etc.
This technique is used for the transfection of
plant and animal cells. In this technique
needle-like nanostructures are synthesized
perpendicularly to the surface of a substrate.
🞂 Recombinant DNA is attached to the
nanostructure surface. A chip with arrays of
these needles is then pressed against cells or
tissue.
Method of Magnetofection:

🞂 Magnetofection, or Magnet assisted trans-

fection is a method, which uses magnetic
force to deliver recombinant DNA into target
host cells. Nucleic acids are first associated
with magnetic nanoparticles. Then,
application of magnetic force drives the
nucleic acid particle complexes towards and
into the target host cells, where the cargo is
released. This has been successfully used to
transfect the plant and animal cells.
Method of Protoplast Fusion:

🞂 This technique is used for introducing gene

of interest into plant and animal cells. In this
technique first we transfer the recombinant
DNA into a bacterial cell then dissolve its cell
wall by treating it with lysozyme.
🞂 After this we fuse the host protoplast with the
bacterial cell (lacking cell wall) by the help of
polyethylene glycol (PEG). The transfected
cells are then selected by suitable methods.
Method of Virus Mediated Gene
Transfer:
🞂 In other way the gene can be packed into a virus and allow it to
infect the host cell without harming it in any way. This method
can be used both for the transformation of prokaryotic host cell
as well as transfection of eukaryotic host cells. In the case of
bacterial host cells the recombinant DNA can be packed into the
empty head of a specially designed bacteriophage (e.g., lambda
phage) and allow the virion to infect the host cell.

🞂 Similarly, while transfecting the plant host cells we can follow the
similar strategy by using plant viruses like Caulimo virus and
Gemini virus. In the case of animals, retrovirus infection of
embryos has been used for the production of transgenic mice.

🞂 This virus has been found to be an efficient vector system for

animals. The virus carrying the gene of interest transfers it into
the genome of embryonic cells leading to its integration and
production of transgenic animals.
 Detection of the
Transgene:
• PCR or Southern blotting of genomic DNA can
detect the foreign DNA in the
blastocyst, embryo or young animal.
• The expression of the transgene can be
estimated by measuring the enzyme
activity coded by the transgene.
• Or by, Western analysis or Enzyme-Linked
Immunosorbent Assay
(ELISA).
Applications of Gene Transfer
in Farm
Animals
First Transgenic Cow Clone for Mastitis
Disease Resistance

“Annie” , born in March 2000

Human Insulin-Like Growth Factor I(IGF-I)
Produced in the MammaryGlands of
Transgenic
Rabbits
CHANGING AMINO ACID REQUIREMENTS
USING ANIMAL
BIOTECHNOLOGY
Wool Production from
Sheep
Transgenic Fish forGrowth
Enhancement
• Recombinant DNA and Genetic engineering
began to be applied to aquacultural species in
1980s.

• Mammalian GH gene constructs are used

to produce a Transgenically growth
enhanced
tilapia fishes.
• GH gene constructs
increased the weight of
tilapia fishes in
approximately a 5
– 11 fold.
Transgenic animal for
pharmaceutical production
❑ the production of transgenic Drosophila by the injection of
plasmid vectors containing P elements into the fly egg.
Transgenic Drosophila provide us with another illustration
of the use of the bacterial lacZ gene as a reporter in the
study of gene regulation during development.

❑ The lacZ gene is fused to the promoter region of a

Drosophila heat shock gene, which is normally activated by
high temperatures. This construct is then used to generate
transgenic flies. Subsequent to heat shock, the flies are
killed and bathed in X-Gal.

❑ The resulting pattern of blue tissues provides information

on the major sites of action of the heat shock gene .
❑ In both these cases, because it is the egg that is
initially made transgenic, the extra DNA can find its
way into germ-line cells, is then passed on to the
progeny derived from these cells, and behaves from
❑ Production of a pharmaceutically
important protein in the milk of
transgenic sheep. The gene of
interest encodes a protein that is of
therapeutic importance, such as
tissue plasminogen activator used to
dissolve blood clots in humans.

❑ The gene is placed under control of

the β-lactoglobulin promoter, active
only in mammary tissue, and is
introduced into sheep ova by
microinjection of the expression
vector into the nucleus.

❑ The injected ova are implanted into

foster mothers, and progeny
expressing the transgene are
identified by PCR amplification of
chromosomal DNA by using primers
from the sequence of the gene of
interest.

❑ Transgenic sheep express this gene

❑ some eukaryotic proteins cannot be easily expressed
in large amounts in bacteria, and eukaryotic
expression systems need to be employed.

❑ A widely used vector–expression system for

eukaryotic proteins is insect baculovirus, into which
genes are inserted and expressed at high rates in
cultured insect cells.
❑ Baculovirus is a very large DNA virus (genome of about 150 kb) that infects
insect cells. To express a foreign gene in baculovirus, the gene of interest is
cloned in place of the viral coat-protein gene in a plasmid carrying a small
part of the viral genome. The recombinant plasmid is cotransfected into
insect cells with wild-type baculovirus DNA.

❑ At a low frequency, the plasmid and viral DNAs recombine through

homologous sequences, resulting in the insertion of the foreign gene into
the viral genome. Virus plaques develop, and the plaques containing
recombinant virus look different because they lack the coat protein. The
plaques with recombinant virus are picked and expanded.

❑ This virus stock is then used to infect a fresh culture of insect cells,
resulting in high expression of the foreign protein.
 What is Plant Tissue Culture?

“… the aseptic culture of plant

protoplasts, cells, tissues or
organs under conditions which
lead to cell multiplication or
regeneration of organs or whole
plants “
Totipotency ….
… each
complete
livinggenetic
cell has a complete
blue print genetic
blueprint and therefore has the potential to
develop into an entire plant.
……cells
cellslines
differentiate
differentiate to form specialised
tissues and organs

… unlike animal
living plant cells,
cells canliving plant cells can
re-differentiate
de-differentiate and then re-differentiate to
form different cell types
Therefore,

tissue can be regenerated from

explants such as cotyledons,
hypocotyls, leaf, ovary, protoplast,
petiole, root, anthers, etc.
Organogenesis:
The process of initiation and
development of a structure that shows
natural organ form and/or function.

Embryogenesis:
The process of initiation and
development of embryos or embryo-like
structures from somatic cells (Somatic
embryogenesis).
 What for?

• Tissue culture produces clones, in

which all product cells have the same
genotype (unless affected by mutation
during culture)
 What for?

• A more recent advance is the use of

plant and animal tissue culture along
with genetic modification using viral and
bacterial vectors and gene guns to
create genetically engineered
organisms
 First commercial use
• The first commercial use of
plant clonal propagation on
artificial media was in the
germination and growth of
orchid plants, in the 1920’s

• In the 1950’s and 60’s there

was a great deal of research,
but it was only after the
development of a reliable
artificial medium (Murashige
& Skoog, 1962) that plant Young cymbidium orchids
tissue culture really ‘took off’
commercially
 What is needed?
Tissue culture has several critical requirements:

• Appropriate tissue (some tissues culture better than

others)

• A suitable growth medium containing energy sources

and inorganic salts to supply cell growth needs. This can
be liquid or semisolid

• Aseptic (sterile) conditions, as microorganisms grow

much more quickly than plant and animal tissue and can
over run a culture
 What is Needed
• Growth regulators - in plants, both auxins
& cytokinins.

• Frequent subculturing to ensure adequate

nutrition and to avoid the build up of waste
metabolites
Culturing Plant Tissue - the steps

• Selection of the plant

tissue (explant) from a
healthy vigorous
‘mother plant’ - this is
often the apical bud,
but can be other tissue

• This tissue must be

sterilized to remove
microbial contaminants
Callus – an undifferentiated tissue
Elimination of microbial contaminants

Surface contaminants - principally microbial saprophytes

that are eliminated by surface disinfestation

Internal contaminants - principally pathogens that are

eliminated by thermotherapy (35-40 C) and culture of
explants free of organisms or by antibiotics

Maintenance of asepsis (free from microorganisms) - during

excision and culture - procedures are carried out in
sterile laminar flow positive pressure hoods (0.3 m
filters)
Common Plant Tissue Disinfestants

Concentration of Time
Agent Active Ingredient Phytotoxicity (min)
Na hypochlorite
(Laundry Bleach) 0.25-1% Moderate 5-20
Ca hypochlorite 9-10% Moderate 5-20
H2O2 3-10% High 5-20
Alcohol
(ethanol or
isopropanol) 70% High 30 sec

These disinfestants can be used in combination and the

effectiveness of these solutions is enhanced by using a wetting agent
such as a detergent.
Culturing Plant Tissue - the steps
• Establishment of the
explant in a culture
medium. The medium
sustains the plant cells and
encourages cell division. It
can be solid or liquid

• Each plant species has

particular medium
requirements that must be
established by trial and
error
 Culture Medium constituents

• Inorganic salt formulations

• Source of carbohydrate
• Vitamins
• Water
• Plant hormones - auxins, cytokinins, GA’s
• Solidifying agents
Composition
of tissue
culture
medium is
complex
• Two Hormones Affect Plant
Differentiation:
– Auxin: Stimulates Root Development
– Cytokinin: Stimulates Shoot Development

• Generally, the ratio of these two

hormones can determine plant
development:
–  Auxin ↓Cytokinin = Root Development
–  Cytokinin ↓Auxin = Shoot Development
– Auxin = Cytokinin = Callus Development
2iP/IAA 0.5/2.5

Effect of
Kin/IAA 0.5/2.5 different
auxine and
Kin/IBA 0.5/2.5 cytokinine
concentration
on tissue
Kin/IBA 0.5/0.5
developemt

Kin/NAA 0.5/0.5
Culturing Plant Tissue - the steps

• The rooted shoots are

potted up (deflasked)
and ‘hardened off’ by
gradually decreasing
the humidity

• This is necessary as
many young tissue
culture plants have no
waxy cuticle to prevent
water loss
Factors Affecting Plant Tissue Culture
• Growth Media
– Minerals, Growth factors, Carbon source, Hormones
• Environmental Factors
– Light, Temperature, Photoperiod, Sterility, Media
• Explant Source
– Usually, the younger, less differentiated the explant,
the better for tissue culture
• Genetics
– Different species show differences in amenability to
tissue culture
– In many cases, different genotypes within a species
will have variable responses to tissue culture;
response to somatic embryogenesis has been
transferred between melon cultivars through sexual
hybridization
Why do Plant Tissue Culture?
• A single explant can be multiplied into several
thousand plants in less than a year - this
allows fast commercial propagation of new
cultivars

• Taking an explant does not usually destroy

the mother plant, so rare and endangered
plants can be cloned safely

• Once established, a plant tissue culture line

can give a continuous supply of young plants
throughout the year
 Why do Plant Tissue Culture? II
• In plants prone to virus diseases, virus free
explants (new meristem tissue is usually virus
free) can be cultivated to provide virus free
plants

• Plant ‘tissue banks’ can be frozen, then

regenerated through tissue culture

• Plant cultures in approved media are easier to

export than are soil-grown plants, as they are
pathogen free and take up little space (most
current plant export is now done in this
manner)
Tissue Culture Applications
- Micropropagation
- Germplasm preservation
- Somaclonal variation & mutation selection
- Embryo Culture
- Haploid & Dihaploid Production
- In vitro hybridization – Protoplast Fusion
- Industrial Products from Cell Cultures
Micropropagation
A single explant can be
multiplied into several
thousand plants in less than
a year - this allows fast
commercial propagation of
new cultivars
Features of Micropropagation
• Clonal reproduction
– Way of maintaining heterozygozity
• Multiplication stage can be recycled many
times to produce an unlimited number of clones
– Routinely used commercially for many ornamental
species, some vegetatively propagated crops
• Easy to manipulate production cycles
– Not limited by field seasons/environmental influences
• Disease-free plants can be produced
– Has been used to eliminate viruses from donor plants
Micropropagation

– generation of genetical
identical plants
 Micropropagation of almost all
the fruit crops and vegetables
is possible now

• Some examples: dwarfing sweet cherry,

Shade trees, Ornamental shrubs, Roses,
Clematis, Lilacs, Saskatoon berries,
Nutraceutical Plants, Rhododendron,
Azalea, mustard, corn, soybeans, wheat,
rice, cotton, tomato, potato, citrus, turf,
legumes
Micropropagation
- allows rapid
propagation of new varieties
- economical in time and space
- disease free

example: violets
 Germplasm Preservation
Slow growth techniques

o e.g.: ↓ Temp., ↓ Light, media supplements

(osmotic inhibitors, growth retardants), tissue
dehydration, etc…
o Medium-term storage (1 to 4 years)
Germplasm Preservation

Example:
Titan Arum
(Amorphophallus titanum)
 Germplasm Preservation

Cryopreservation
o Ultra low temperatures (-196 °C)
o Stops cell division &
metabolic processes
o Very long-term (indefinite?)
Cryopreservation Requirements
• Storage
– Usually in liquid nitrogen (-196oC) to avoid changes in
ice crystals that occur above -100oC
• Thawing
– Usually rapid thawing to avoid damage from ice crystal
growth
• Recovery
- Thawed cells must be washed of cryoprotectants
and nursed back to normal growth
– Avoid callus production to maintain genetic stability
Cryopreservation Requirements
• Preculturing
– Usually a rapid growth rate to create cells with small
vacuoles and low water content
• Cryoprotection
– Glycerol, DMSO, PEG, etc…, to protect against ice
damage and alter the form of ice crystals
• Freezing
– The most critical phase; one of two methods:
• Slow freezing allows for cytoplasmic dehydration
• Quick freezing results in fast intercellular freezing with little
dehydration
 Embryo Culture
Embryo culture developed from the need
to rescue embryos (embryo rescue)
from wide crosses where fertilization
occurred, but embryo development did
not occur
 Embryo Culture Uses
• Rescue F1 hybrid from a wide cross
• Overcome seed dormancy, usually with
addition of hormone to media (GA)
• To overcome immaturity in seed
– To speed generations in a breeding program
– To rescue a cross or self (valuable genotype) from
dead or dying plant
 Embryo Culture Uses

Rescue F1 hybrid from a wide cross

Example: Anthurium
 Embryo Rescue Process
• Make cross between two species
• Dissect embryo (usually immature)
– The younger the embryo, the more difficult to culture
• Grow on culture medium using basic tissue culture
techniques, use for breeding if fertile
• Many times, resulting plants will be haploid because
of lack of pairing between the chromosomes of the
different species
– This can be overcome by doubling the chromosomes,
creating allotetraploids
 Embryo rescue process

15 days

30 days

50 days 80 days
Regeneration of grape plants via
somatic embryosgenesis
Potential uses for tissue culture
in plant breeding
• Eliminate virus from infected plant
selection

– Either via meristem culture or sometimes via

heat treatment of cultured tissue (or
combination)
 Phytosanitation

Bacteria or Virus
infected plant
Infection of shoot Regeneration of infected
meristem plants

In a number of plants the

shoot meristem doe‘s not Regeneration of healthy
get infected pathogen-free plants
Eliminate virus from infected plant selection.

often used for potato, strawberry, banana, citrus

Isolation of the shoot meristem

 Somaclonal Variation

• There are two general types of Somaclonal Variation:

– Heritable, genetic changes (alter the DNA)
– Stable, but non-heritable changes (alter gene
expression, epigenetic)

– used in mutation breeding

Somaclonal Breeding Procedures
• Use plant cultures as starting material
– Idea is to target single cells in multi-cellular culture
– Usually suspension culture, but callus culture can work
(want as much contact with selective agent as possible)
• Optional: apply physical or chemical mutagen
• Apply selection pressure to culture
– Target: very high kill rate, you want very few cells to
survive, so long as selection is effective
• Regenerate whole plants from surviving cells
Somaclonal/Mutation Breeding
• Advantages
– Screen very high populations (cell based)
– Can apply selection to single cells
• Disadvantages
– Many mutations are non-heritable
– Requires dominant mutation (or double recessive
mutation); most mutations are recessive
Industrial Products from Cell Cultures

• Secondary metabolites produced by plants

– Alkaloids, Terpenoids, Steroids, Anthocyanins,
Polyphenols
• Often unclear function in the plant
• Often restricted production (specific species,
tissue or organ)
• Many are commercially valuable
• Cell culture techniques allow large-scale
production of specific secondary metabolites
Animal Cell Culture
• Cell culture refers to the process by which cells are grown in a
controlled artificial environment. Cells can be maintained in vitro
outside of their original body by this process which is quite simple
compared to organ and tissue culture.
• In a cell culture technique, cells are removed from an animal or a plant
and grown subsequently in a favorable environment. For animal cell
culture the cells are taken from the organ of an experimental animal.
The cells may be removed directly or by mechanical or enzymatic
action. Examples of cells used to culture are fibroblast, lymphocytes,
cells from cardiac and skeletal tissues, cells from liver, breast, skin,
and kidney and different types of tumor cells.
 How Are Cell Cultures Obtained? (Primary Culture)

When cells are surgically removed from an organism and placed into a suitable
culture environment, they will attach, divide and grow. This is called a Primary
Culture.

There are two basic methods for doing this:

First: Explant Cultures, small pieces of tissue are attached to a glass or treated
plastic culture vessel and bathed in culture medium. After a few days, individual
cells will move from the tissue explant out onto the culture vessel surface or
substrate where they will begin to divide and grow.
• The second: More widely used method, speeds up this process
by adding digesting (proteolytic) enzymes, such as trypsin or
collagenase, to the tissue fragments to dissolve the cement
holding the cells together. This creates a suspension of single
cells that are then placed into culture vessels containing culture
medium and allowed to grow and divide. This method is called
Enzymatic Dissociation.
 Secondary cell culture and cell line

• When a primary culture is sub-cultured, it is known as

secondary culture or cell line or sub-clone. The process
involves removing the growth media and disassociating the
adhered cells (usually enzymatically).
• Sub-culturing of primary cells to different divisions leads
to the generation of cell lines. During the passage, cells
with the highest growth capacity predominate, resulting in
a degree of genotypic and phenotypic uniformity in the
population. However, as they are sub-cultured serially, they
become different from the original cell.
• Cell lines are categorized into two types:
• Finite cell lines
• The cell lines which go through a limited number of cell
division having a limited life span are known as finite cell
lines. The cells passage several times and then lose their
ability to proliferate, which is a genetically determined
event known as senescence. Cell lines derived from
primary cultures of normal cells are finite cell lines.
• Continuous cell lines
• When a finite cell line undergoes transformation and acquires the
ability to divide indefinitely, it becomes a continuous cell line. Such
transformation/mutation can occur spontaneously or can be chemically
or virally induced or from the establishment of cell cultures from
malignant tissue. Cell cultures prepared in this way can be sub-cultured
and grown indefinitely as permanent cell lines and are immortal.
• These cells are less adherent, fast growing, less fastidious in their
nutritional requirements, able to grow up to higher cell density and
different in phenotypes from the original tissue. Such cells grow more
in suspension. They also have a tendency to grow on top of each other
in multilayers on culture-vessel surfaces.
 Cell Culture Systems

Two basic culture systems are used for growing cells. These are based primarily
upon the ability of the cells to either grow attached to a glass or treated
plastic substrate (Monolayer Culture Systems) or floating free in the
culture medium (Suspension Culture Systems).

Monolayer cultures are usually grown in tissue culture treated dishes, T-flasks,
roller bottles, or multiple well plates, the choice being based on the number
of cells needed, the nature of the culture environment, cost and personal
preference.

Suspension cultures are usually grown either:

1. In magnetically rotated spinner flasks or shaken Erlenmeyer flasks where
the cells are kept actively suspended in the medium.

2. In stationary culture vessels such as T-flasks and bottles where, although the
cells are not kept agitated, they are unable to attach firmly to the substrate.
 Cultivation Methods of animal cells

Many cell lines, especially those derived from normal tissues, are
considered to be Anchorage-Dependent, that is, they can only grow
when attached to a suitable substrate.

Anchorage dependent culture

Anchorage dependent cell lines can be grown by the following
methods:

A - Conventional methods
They include MD bottles, T flasks, Roux bottles and Rollers
B - New trends ( microcarrier cultures) :

(1) Microcarriers: These are bead-like structures ranging from 100-

180 u in diameter that can be held in homogeneous suspension in
stirred reactors. They are capable of providing a large surface area
to cells in small volumes of media. Commercially available
microcarriers have been made from a variety of materials such as
DEAE Sephadex, cellulose, glass, gelatin etc.

(2) These type of microcarriers will produce better yields, the cells
growing in them are not exposed to shearing forces in fermentors.
Such microcarriers have been used for cultivating different cells
and also for viruses.
 Advantages of microcarrier cultures:

1-High surface area to volume ratio can leads to high cell densities
per unit volume with a potential for obtaining highly
concentrated cell products .

2-Cell propagation can be carried out in a single high productivity

vessel instead of using many low productivity units .

3-Cell sampling is easy, since the beads settle down easily, cell
harvesting and downstream processing of products is easy .
 Applications of Microcarrier Cultures

-An excellent tool for studying different aspects of cell biology (such as cell-
to-cell or cell-to-substrat interactions).

-Cultivation of invertebrates cells for the production of immunologicals like

interferons, interleukins, growth factors etc.

-Cell differentiation and maturation.

-Metabolic studies (such cells can also be used for electron microscopic
examinations or for the isolation of cell organelles such as the cell membrane).

-Microcarrier beads confluent with allogenic tumour cells can be injected in

mice to increase humoral and cell-mediated immunity.
 Applications of Animal Cell and Tissue Culture

1-Model Systems
Animal cell cultures provide a good model system for studying:
-Basic cell biology and biochemistry.
-The interactions between disease-causing agents and cells.
-The effects of drugs on cells.
- The process and triggers for aging.
- Nutritional studies.

2-Toxicity Testing
Animal cultured cells are widely used alone or in
conjunction with animal tests to study the effects of new
drugs, cosmetics and chemicals on survival and growth in a
wide variety of cell types. Especially important are liver-
and kidney-derived cell cultures.
 Live cell
Dead
cell
 3-Cancer Research

Since both normal cells and cancer cells can be grown in culture, the basic
differences between them can be closely studied. In addition, it is possible, by
the use of chemicals, viruses and radiation, to convert normal cultured cells to
cancer causing cells. Thus, the mechanisms that cause the change can be
studied.

Cultured cancer cells also serve as a test system to determine suitable drugs
and methods for selectively destroying types of cancer.
 4-Virology

One of the earliest and major uses of cell culture is the

replication of viruses in cell cultures (in place of animals)
for use in vaccine production.

Animal cell cultures are also widely used in the clinical

detection and isolation of viruses, as well as basic research
into how they grow and infect organisms.
5-Genetic Engineering
Production of commercial proteins, large scale
production of viruses for use in vaccine
production e.g. polio, rabies, chicken pox,
hepatitis B & measles
6-Gene therapy
Cells having a functional gene can be replaced
to cells which are having non-functional gene
Transgenic plants in agriculture

Prof. Reda E.A. Moghaieb

 Conventional
breeding

Tissue culture Plants

Genetic engineering
 Plant Transformation

• Plants are the easiest of higher organisms to transform

• Both physical and biological methods exist for

transformation

• Until recently, only transgenic organisms in wide public

release were plants
 Transgenic Plants

• Currently 16 countries permit the cultivation of transgenic crops

• 99% of the total acreage is in 6 countries

– USA, China, Argentina, Canada, Brazil* & Australia

• Five crops are currently in release

– soybean, cotton, corn, canola, squash

– tomato, potato and flax in past
 Transgenic Plants

• Since the first widespread release in 1996, land devoted

to transgenic crops has increased by 10 - 50% yearly

• 90% of Canadian canola is GMO - mostly transgenic

• 20+ countries suspected of growing transgenic crops

without official approval
 Plant Transformation Methods

Physical Chemical Biological In planta

Microinjection A. Tumefaciens
Pressure PEG
A. Rhizogenes
Biolistics - gene gun/ DEAE-dextran
particle bombardment Calcium
Virus-mediated
Electroporation phosphate
Microinjection Artificial lipids
Silica/carbon fibers Proteins
Lazer mediated Dendrimers
 Biological Transformation

- Agrobacterium tumefaciens &

Agrobacterium rhizogenes
Recombinant DNA technology and
crop improvement

-Allows utilization of every species

-Allows direct transfer of a single gene

- Requires a method of gene transfer into plant cells

-Requires regenerable plant cells

Totipotency of many plant cell types allows for

regeneration of entire plants from single cells
Whole plants can be regenerated via cell and tissue
culture
Somatic
embryogenesis
 Agrobacterium
A unique bacterial species
Plant-Fungal-Animal Transformation
 Agrobacterium

Agrobacterium (disease symptomology and host

range)

A. radiobacter - “avirulent” species

A. tumefaciens - crown gall disease

A. rhizogenes - hairy root disease

A. rubi - cane gall disease

A. vitis - galls on grape and a few

other plant species
Otten et al., 1984
Agrobacterium-Plant Interactions

Crown gall on
tomato stem
 Agrobacterium tumefaciens
1. Soil bacterium closely related to Rhizobium.
2. Causes crown gall disease in plants (dicots).
3. Infects at root crown or just below the soil line.

4. Can survive independent of plant host in the soil.

5. Infects plants through breaks or wounds.

6. Common disease of woody shrubs, herbaceous plants,

particularly problamatic with many members of the rose
family.

7. Galls are spherical wart-like structures similar to tumors.

 Only known natural example of DNA
transport between Kingdoms

1. (Virulent) strains of A.
tumefaciens contain a
200-kb tumor inducing
(Ti) plasmid

2. Bacteria transfer a
portion of the plasmid T-DNA →
DNA into the plant
host (T-DNA).
 The T-DNA is transferred from the
Bacteria into the Nucleus of the Plant
1. Stably integrates (randomly) into the plant genome.

2. Expression of genes in wild-type T-DNA results in

dramatic physiological changes to the plant cell.

3. → Synthesis of plant growth hormones (auxins and

cytokinins) → neoplastic growth (tumor formation)
 Opine Biosynthesis

1. Within tumor tissues, the synthesis of various unusual

amino acid-like compounds are directed by genes
encoded on the integrated plasmid.

2. The type of opine produced is specified by the bacterial

T-DNA

3. Opines are used by the bacteria as a carbon (nutrient)

source for growth.

4. Opine catabolism within bacteria is mediated by genes

encoded on the Ti plasmid.
 How is the signal recognition
(acetosyringone and other plant phenolics)
converted to gene activation and other
cellular responses?
Plant-Derived Phenolics Initiate T-DNA Transfer

A - acetosyringone D - ethyl ferulate

B - coniferyl alcohol E - bromoacetosyringone
C - coniferin
 Plant-Derived Sugars Also Play a
Role in Virulence

• Galactose, glucose, arabinose, other aldose

sugars induce vir gene expression.

• Mutations in chvE abolish the action of

sugars.

• ChvE is a periplasmic sugar-binding protein

that functions in chemotaxis (swarm assays).

• These sugars synergistically enhance the

stimulatory effects of AS in lacZ reporter
assays.

• The periplasmic domain of VirA is required

for synergism with ChvE.
• Acetosyringone acts through VirA located in the
bacterial membrane, probably by direct binding.
• VirA undergoes autophosphorylation (as a dimer?) in
response to acetosyringone.
• VirG is the response component of the VirA/VirG
complex.
• VirG is a transcriptional inducer of other vir genes
governing T-DNA transfer and integration.
• VirG activity requires phosphorylation via transfer of
a phosphate group from VirA.
VirB – type IV secretion system (pilus) for T-
DNA transfer

VirD1 – nicking of T-DNA borders

VirD2 – Endonuclease; chaperone to plant nucleus;

integration into host genome

VirE2 – Single strand binding protein; formation of T-complex;

nuclear targeting and import

T-DNA Encodes:

• Auxin and cytokinin biosynthetic genes

• Opine biosynthesis
 Agrobacterium/plant
interactions
Agrobacterium at
wound site transfers
T-DNA to plant cell.

opines
Agrobacterium in soil use
opines as nutrients.
Overview of the Infection Process
Genes required to breakdown opines for use as a nutrient
source are harbored on the Ti plasmid in addition to vir
genes essential for the excision and transport of the T-
DNA to the wounded plant cell.

T-DNA 23 kb

tra bacterial
conjugation
pTi
vir genes ~200 kb
for transfer to
the plant opine catabolism
 Ti Plasmid
1. Large (200-kb)
2. Conjugative
3. ~10% of plasmid transferred to plant cell
after infection
4. Transferred DNA (called T-DNA) integrates
semi-randomly into nuclear DNA
5. Ti plasmid also encodes:
– enzymes involved in opine metabolism
– proteins involved in mobilizing T-DNA (Vir
genes)
 T-DNA

LB auxA auxB cyt ocs RB

LB, RB – left and right borders (direct repeat)

auxA + auxB – enzymes that produce auxin
cyt – enzyme that produces cytokinin
Ocs – octopine synthase, produces octopine

These genes have typical eukaryotic expression signals!

 Ti plasmids can be classified according to
the opines produced

1. Nopaline plasmids: carry gene for synthesizing

nopaline in the plant and for utilization
(catabolism) in the bacteria. Tumors can
differentiate into shooty masses (teratomas).

2. Octopine plasmids: carry genes(3 required) to

synthesize octopine in the plant and catabolism in
the bacteria. Tumors do not differentiate, but
remain as callus tissue.
3. Agropine plasmids: carry genes for agropine synthesis
and catabolism. Tumors do not differentiate and die out.

(Nopaline)
H2N
CNH(CH2)2CHCO2H
HN NH
HO2C(CH2)2CHCO2H
 Ti plasmids and the bacterial chromosome
act in concert to transform the plant

1. Agrobacterium tumefaciens chromosomal

genes: chvA, chvB, pscA required for initial
binding of the bacterium to the plant cell and code
for polysaccharide on bacterial cell surface.

2. Virulence region (vir) carried on pTi, but not in

the transferred region (T-DNA). Genes code for
proteins that prepare the T-DNA and the bacterium
for transfer.
3. T-DNA encodes genes for opine synthesis and
for tumor production.

4. occ (opine catabolism) genes carried on the pTi

and allows the bacterium to utilize opines as
nutrient.
 Agrobacterium chromosomal DNA

pscA

chvA chvB
T-DNA-inserts into plant genome

tra bacterial
for transfer pTi conjugation
to the plant vir genes
opine catabolism
oriV
 Generation of the T-strand

Left Border Right

Border
T-DNA

overdrive

5’
virD/virC

VirD nicks the lower strand (T-strand) at the right

border sequence and binds to the 5’ end.
 Generation of the T-strand

Left Right
border T-DNA border

gap filled in
virE T-strand
D
virD/virC
1. Helicases unwind the T-strand which is
then coated by the virE protein.

2. ~one T-strand produced per cell.

 Left Right
border T-DNA border
D
T-strand coated with virE

virD nicks at Left Border sequence

1. Transfer to plant cell.

2. Second strand synthesis
3. Integration into plant chromosome
The vir region is responsible for the transfer of T-DNA
to the wounded plant cell.
virA is the sensor.
activated virG
membrane

constitutive

virA virG
positive Note: activated virG causes
receptor for regulator its own promoter to have a
acetyl- for other new start point with
syringone vir genes increased activity.
 Asg virA is the sensor.
1 2
P
virA Asg
triggers auto-
bacterial
phosphorylation of
membrane
Acetylsyringone is virA
produced by wounded
plant cells (phenolic
compound). 3 virG activates
P transcription
VirA phosphorylates from other vir
virG which causes virG virG promoters.
to become activated.
virG is the effector.
The vir region is responsible for the transfer of T-DNA
to the wounded plant cell.
sensor effector

virA virG virD virE

virB
virC ssDNA
membrane Binds endo- binding
protein; ATP- overdrive nuclease protein.
binding DNA. nicks T- Binds T-
DNA strand.

Note: The virA-virG system is related to the EnzZ-OmpR

system that responds to osmolarity in other bacteria.
Common Transformation Protocols

1. Leaf-disc transformation - after selection and

regeneration with tissue culture, get plants
with the introduced gene in every cell

2. Floral Dip – does not require tissue culture.

Reproductive tissue is transformed and the
resulting seeds are screened for drug-
resistant growth. (Clough and Bent (1998) Floral dip: a
simplified method for Agrobacterium-mediated transformation of
Arabidopsis thaliana. Plant Journal 16, 735–743)
Agrobacterium transformation
 Problems with the use of Agrobacterium for transformation:

1. Same DNA between T-DNA borders can be inserted into

multiple chromosomal regions of the transformed plants. Easy
to get as many as 10 copies inserted during a single
transformation. Makes generating of homozygous plant
difficult.

2. Only able to transform dicotyledonous plants with sufficient

efficiency. Attempts to expand the host range of bacterium has
met with little success.
Agrobacterium rhizogenes
 A. rhizogenes, the causative agent of hairy root
syndrome, is a common soil bacterium (Gram negative)
capable of entering a plant through a wound and causing
a proliferation of secondary roots. The underlying
mechanism of hairy root formation is the transfer of
several bacterial genes to the plant genome. The
observed morphogenic effects in the plants after
infection have been attributed to the transfer of part of a
large plasmid known as the Ri (root-inducing) plasmid.
The symptoms observed with A.rhizogenes are
suggestive of auxin effects resulting from an increase in
cellular auxin sensitivity rather than auxin production.
 Ri plasmids
• Ri plasmids are large (200 to greater than 800 kb) and contain
one or two regions of T-DNA and a vir (virulence) region, all of
which are necessary for tumorgenesis.
• The Ri-plasmids are grouped into two main classes according to
the opines synthesized by hairy roots:

1- First, agropine-type strains induce roots to synthesise agropine,

mannopine and the related acids.

2-Second, mannopine-type strains induce roots to produce

mannopine and the corresponding acids. The agropine-type Ri-
plasmids are very similar as a group and a quite distinct group from
the mannopine-type plasmids. Perhaps the most studied Ri-plasmids
are agropine-type strains, which are considered to be the most
virulent and therefore more often used in the establishment of hairy
root cultures.
The genes responsible for hairy root formation

• The T-DNA of the agropine-type Ri-plasmid consists of two

separate T-DNA regions designed the TL-DNA and TR-DNA.
Each of the T-DNA fragments spans a 15 - 20 kb region, and
they are separated from each other by at least 15 kb of non-
integrated plasmid DNA.
• These two fragments can be transferred independently during
the infection process. The genes encoding auxin synthesis (tms1
and tms2) and agropine synthesis (ags) have been localised on
the TR-DNA of the agropine type Ri-plasmid.

• The mannopine type Ri-plasmids contain only one T-DNA that

shares considerable DNA sequence homology with TL of the
agropine-type plasmids.
Mutation analysis of the TL-DNA has led to
identification of four genetic loci, designed locus
rolA, rolB, rolC, and rolD, which affect hairy root
induction. The complete nucleotide sequence of the
TL-region revealed the presence of 18 open-
reading frames (ORFs), 4 of which, ORFs 10, 11,
12 and 15, respectively, correspond to the rolA,
rolB, rolC, and rolD loci.
• It was also shown that rolA, rolB, and rolC play the most important
role in hairy root induction. In particular, rolB seems to be the most
crucial in the differentiation process of transformed cells, while rolA
and rolC provide with accessory functions. rolA is associated with
internode shortening and leaf wrinkling; rolB is responsible for
protruding stigmas and reduced length of stamens; rolC causes
internode shortening and reduced apical dominance.

• Although the TR-DNA is not essential for hairy root formation it

has been shown that the aux1 gene harbored in this segment
provides to the trasformed cells with an additional source of auxin.
 Advantages:

Agrobacterium rhizogenes-mediated transformation,

also known as hairy root transformation, offers several
advantages for genetic modification of plants. Here
are some of the key advantages:
1. High transformation efficiency: Agrobacterium rhizogenes has a
high transformation efficiency, meaning it can effectively deliver
and integrate foreign genes into the plant genome. This allows
for the generation of a large number of transformed plants,
increasing the chances of obtaining desired traits.

2. Rapid transformation and growth: Hairy roots induced by

Agrobacterium rhizogenes grow rapidly and can be easily
propagated in vitro. This enables faster generation of transformed
plants compared to other transformation methods.
3. Ease of selection: Agrobacterium rhizogenes usually
carries a selectable marker gene, such as antibiotic resistance,
along with the gene of interest. This allows for easy selection of
transformed plants by applying the appropriate selection pressure,
such as antibiotic treatment.

4. Production of secondary metabolites: Hairy roots are unique

in their genetic and biosynthetic stability. Their fast growth, low
doubling time, ease of maintenance, and ability to synthesize a
range of chemical compounds offers an additional advantage as a
continuous source for the production of valuable secondary
metabolites. hairy roots often exhibit enhanced production of
secondary metabolites, including various valuable compounds such
as alkaloids, flavonoids, and terpenoids. This makes it a useful tool
for the production of pharmaceuticals, flavors, and fragrances.
5. Phenotypic stability: Hairy roots generated through
Agrobacterium rhizogenes-mediated transformation generally
display stable phenotypes over multiple generations. This
stability is advantageous for studying gene function and
evaluating the effects of gene expression on plant traits.

6. Compatibility with a wide range of plant species:

Agrobacterium rhizogenes can infect and transform a broad
range of plant species, including both dicots and monocots.
This versatility makes it a valuable tool for genetic
engineering in diverse plant species.
7-Plant regeneration
• Transformed roots are able to regenerate whole viable plants;
hairy roots as well as the plants regenerated from hairy roots are
genetically stable. However, in some instances transgenic plants
have shown an altered phenotype compared to controls.
• Plants can be regenerated from hairy root cultures either
spontaneously (directly from roots) or by transferring roots to
hormone-containing medium. The advantage of Ri plasmid-based
gene transfer is that spontaneous shoot regeneration is obtained
avoiding the callus phase and somaclonal variations. Ri plasmid-
based gene transfer also has a higher rate of transformation and
regeneration of transgenic plants; transgenic plants can be obtained
without a selection agent thereby avoiding the use of chemicals that
inhibit shoot regeneration; high rate of co-transfer of genes on
binary vector can occur without selection.
8- Agrobacterium tumefaciens mediated transformation
results in high a frequency of escapes; whereas Agrobacterium
rhizogenes mediated transformation consistently yields only
transformed cells that can be obtained after several cycles of
root tip cultures.
• 9). Tree improvement

• A major limitation of tree improvement programs is their

long generation cycle. Classical breeding programs in trees are
slow and tedious and it is difficult to introduce specific genes for
genetic manipulation by crossing parental lines. Agrobacterium
rhizogenes mediated transformation can be a useful alternative,
as a rapid and direct route for introduction and expression of
specific traits. The ability to manipulate tree species at cellular
and molecular level shows great potential and in vitro
transformation and regeneration from hairy roots facilitates
application of biotechnology to tree species. This significantly
reduces the time necessary for tree improvement and gives rise
to new gene combinations that cannot be obtained using
traditional breeding methods. In some tree species root initiation
limits vegetative propagation; by using A. rhizogenes rooting of
cuttings from recalcitrant woody species have been improved.
• 10). Genetic manipulation
Transformed roots provide a promising alternative for the
biotechnological exploitation of plant cells. A. rhizogenes
mediated transformation of plants may be used in a manner
analogous to the well-known procedures employing A.
tumefaciens. A. rhizogenes mediated transformation has also been
used to produce transgenic hairy root cultures and plantlets have
been regenerated. With the exception of the border sequences,
none of the other T-DNA sequences are required for the transfer.
The rest of the T-DNA can be replaced with the foreign DNA and
introduced into cells from which whole plants can be regenerated.
These foreign DNA sequences are stably inherited in a Mendelian
manner. The A. rhizogenes mediated transformation has the
advantage that any foreign gene of interest placed in binary vector
can be transferred to the transformed hairy root clone.
Chloroplast Transformation

Prof. Reda E.A. Moghaieb

• Chloroplast transformation for transgene containment

Chloroplasts : - class of plastids

- organelle
- from cyanobacteria (blue-green bacteria )
- contain chlorophyll
 Transformation of the chloroplast

➢ 1988 : - putting the foreign genes into chloroplast genome

➢ Late 1990 : several biotech companies have initiated

major programs on chloroplast transformation

➢ 1998 : Chloroplast transformation has been touted at least

as far back as 1998 as a means of “containing”
transgenes; that is, preventing them from transferring to
non-GM crops or wild relatives through pollen, and hence
preventing the creation of transgenic herbicide tolerant
weeds. The theory is that chloroplasts are inherited
exclusively through the female line.
 rbcL

trnI-trnA

trnI-trnA
 Nuclear Transformation vector

TERMINATOR/
MONOCISTRONIC POLY A
UNIT ESSENTIAL
HETEROLOGOUS
RANDOM PROMOTER/UTRs
INTEGRATION

35S Selectable marker T 35S Gene of Interest T

Plasmid Backbone
 Chloroplast Transformation vector

TERMINATOR
POLYCISTRONIC NOT
UNIT IMPORTANT NATIVE
SITE PROMOTER/
DIRECTED HETEROLOGOUS
INTEGRATION UTRs

Plasmid Backbone
Cp DNA P 5’UTR Selectable marker 3’UTR 5’UTR Selectable marker 3’UTR Cp DNA

Plasmid Backbone
• Leaf discs are bombarded with plasmid constructs
containing a selectable antibiotic resistance
marker physically linked to the gene of interest,
flanked by DNA for inserting into the correct site
of the chloroplast genome.

• The antibiotic resistance marker most frequently

used is the aadA gene encoding resistance for
spectinomycin and streptomycin, driven by the
promoter of the chloroplast encoded 16S rRNA
gene.

• This transformation procedure applied to tobacco,

Arabidopsis or oil seed rape, generates plants in
which all the chloroplast genomes are uniformly
transformed (a condition referred to as
homoplasmic), despite the fact that tobacco leaf
cells may contain 100 chloroplasts, each
containing 100 copies of the chloroplast genome.
• Transformation of the chloroplast genome by bombarding tobacco
leaves with microprojectiles coated with DNA. Following
bombardment, leaf discs are placed onto antibiotic-containing
medium (panel A). Transgenic plants are regenerated from the
transformed tissue that is able to develop green chloroplasts (panel
B)
In vitro selection and regeneration of chloroplast transgenic tobacco
plants

Confirmation of transgene integration in tobacco.

Fluorescence microscopy
for the identification of
green fluorescent protein
(gfp) in transgenic tobacco
leaves.

A. Non-transformed
tobacco leaf.

B-C. Transformed tobacco

leaves.
Confocal scanning microscopic
confirmation for the localization
of green fluorescent protein
(gfp) in transgenic tobacco
plastids
 Benefits of Chloroplast transformation

• 1. Risk of transgene escape

- Chloroplast genome is maternally inherited and there is rare
occurrence of pollen transmission. It provides a strong level of
biological containment and thus reduces the escape transgene from
one cell to other.
•
2. Expression level
- It exhibits higher level of transgene expression and thus higher
level of protein production due to the presence of multiple copies
of chloroplast transgenes per cell and - Remains unaffected by
phenomenon such as pre or post-transcriptional silencing.
• 3. Homologous recombination
- Chloroplast transformation involves homologous
recombination and is therefore precise and predictable. - This
minimizes the insertion of unnecessary DNA that accompanies
in nuclear genome transformation. - This also avoids the
deletions and rearrangements of transgene DNA, and host
genome DNA at the site of insertion.

• 4. Gene silencing/ RNA interference

- Gene silencing or RNA interference does not occur in
genetically engineered chloroplasts.

• 5. Position effect
- Absence of position effect due to lack of a compact chromatin
structure and efficient transgene integration by homologous
recombination. - Avoids inadvertent inactivation of host gene
by transgene integration.
• 6. Disulphide bond formation
- Ability to form disulfide bonds and folding human proteins results
in high-level production of biopharmaceuticals in plants.

• 7. Multiple gene expression

Multiple transgene expression is possible due to polycistronic
mRNA transcription.

• 8. Expression of edible vaccine

- High level of expression and engineering foreign genes without the
use of antibiotic resistant genes makes this compartment ideal for the
development of edible vaccines.
• 9. Codon usage
- Chloroplast is originated from cyanobacteria through
endosymbiosis. It shows significant similarities with the
bacterial genome. Thus, any bacterial genome can be inserted in
chloroplast genome.
•
10. Expression of toxic proteins
- Foreign proteins observed to be toxic in the cytosol are non-
toxic when accumulated within transgenic chloroplasts as they
are compartmentalized inside chloroplast.

• 11 Environmental biosafety: Chloroplast transformation offers

an additional level of biosafety compared to nuclear
transformation. Since chloroplasts are maternally inherited in
most plants, the spread of transgenes through pollen is
minimized, reducing the risk of gene flow to related plant
species or wild populations
• 12-Increased crop yield and stress tolerance: Chloroplast
transformation can be used to introduce genes that improve
photosynthetic efficiency, enhance stress tolerance (e.g., drought,
salinity, or temperature), or increase crop yield. For example, genes
encoding enzymes involved in carbon fixation or stress-responsive
proteins can be introduced into chloroplasts to enhance plant
productivity and resilience.

• 13 Genetic engineering of algae: Chloroplast transformation is

also applicable to algae, which are photosynthetic
microorganisms. Algal chloroplasts can be engineered to produce
biofuels, high-value metabolites, or enhance their growth
characteristics for bioremediation purposes.
 Multigene Maternal
Engineering Inheritance

High CROP
Advantages of Chloroplast Gene
expression Transformation Containment

IMPROVEMENT
No Vector No Gene
Sequences Silencing
Application of transgenic
plants
 Bacillus thurengiensis
• Gram positive bacterium
• Discovered in Japan in 1902
• Synthetizes insecticidal crystaline proteins (Cry)
• >90 genes encode Cry subunits (-endotoxins)
• 90% of bioinsecticides are from B.t.
• Only 2% of the global pesticide market
• Low field persistance
 Bt-resistance to colorado potato
beetle

Leptinotarsa decemlineata (Say)

NewLeaf potatoes produce the protein toxin in all green tissues and beetle
mortality is essentially 100%. To slow down the development of resistance to
this protein toxin, commercial growers must agree to plant at least 20% of their
potato acreage to standard varieties and use conventional insecticides for
Colorado potato beetle control on this 20%.
http://www3.extension.umn.edu/vegipm/vegpest/cpb.htm#intro
 Virus resistance
• Constructs including genes from the virus
– Coat protein, viral replicase and others
• Different resistance mechanisms
– Effect of viral protein
– RNA degrading mechanism
• Posttranscriptional transgene silencing
Golden rice – inserting a pathway
◼ Synthesis of -carotene from
geranylgeranyl diphosphate requires 3
enzymes
◼ Phytoene synthase (psy)
◼ Phytoene desaturase (crtI)
◼ Synthesis of lycopene (red colour)

◼ Lycopene -cyclase (lcy)

◼ Synthesis of -carotene (yellow colour)
 Golden rice - phenotypes

Untransformed control
2 pathway genes – some
enzymes in the pathway
are expressed in
untransformed rice
3 pathway genes
Fig. 2. Phenotypes of transgenic rice seeds. Bar, 1 cm. (A)
Panel 1, untransformed control; panels 2 through
4, pB19hpc single transformants lines h11a (panel 2),
h15b (panel 3), h6 (panel 4). (B) pZPsC/pZLcyH co-
transformants lines z5 (panel 1), z11b (panel 2), z4a
(panel 3), z18 (panel 4).
Golden rice contains increased levels of pro-vitamin A .

Traditional rice is white (a).

The prototype of golden rice was developed in 2000 and is a light yellow
color (b). It contains 1.6 mg/g of carotenoid.

In 2005, new transgenic lines were developed that dramatically increased

the amount of carotenoid synthesized, making the rice a deep golden color
(c).

This latest form contains 37 mg/g of carotenoid, of which 84% is b-carotene

– trial
Medical hypothesis, 2006

Tomatoes comes in many varieties, colors and shapes

Transgenic tomatoes - expressing different malarial antigens
 Current Opinion in Plant Biology 2007,
10:283–28

Normal and mutant tomato fruit

high-pigment 1 (hp1/hp1), high-pigment 2 (hp2/hp2), Never-ripe (Nr/Nr),

Green-ripe (Gr/Gr), Colorless non-ripening (Cnr/Cnr) &
ripening-inhibitor (rin/rin) mutations
Delivery of a corn-based edible
vaccine

Transgenic corn kernels (a)

Corn snack (b) or

Embryo or germ cells (c)

 Tearless Onion

Dr Eady
Crop & Food Research in New
Zealand and his collaborators in
Japan

As onions are sliced, cells are broken, alliinases - break down aa

sulphoxides - generate sulphenic acids - unstable - rearrange into a volatile
gas - syn-propanethial-S-oxide – diffuses by air - reaches the eye - reacts
with the water to form a diluted solution of sulphuric acid - Tear glands
produce tears to dilute and flush out the irritant
http://www.dailymail.co.uk/news/article-514799/The-orange-purple-
green-cauliflowers-scientists-claim-healthier-you.html
 Purple tomatoes high in anthocyanins
http://news.bbc.co.uk/2/hi/health/768
8310.stm

High anthocyanin purple tomato and

red wild-type tomato
Anthocyanins offer protection against certain cancers,
cardiovascular disease and age-related degenerative diseases.
Anthocyanins also have anti-inflammatory activity, promote
visual acuity and hinder obesity and diabetes.

Tomatoes already contain high levels of the antioxidant

lycopene. Highly processed tomatoes are the best source, or
tomatoes cooked in a little oil, which helps to release the
lycopene from cells.

Flavonoids meanwhile are soluble in water, and foods

containing both water soluble and fat-dissolved antioxidants
are considered to offer the best protection against disease.

In this study the scientists expressed two genes from

snapdragon that induce the production of anthocyanins in
snapdragon flowers. The genes were turned on in tomato
fruit.

Anthocyanins accumulated in tomatoes at higher levels

than anything previously reported for metabolic engineering
in both the peel and flesh of the fruit. The fruit are an intense
purple colour.
World's First Blue Roses On Display In Japan

Tokyo, Japan - World's first blue roses have been

unveiled to the public for the first time at an
international flower fair in Japan, following nearly
two decades of scientific research.
The blue-hued blooms are genetically modified and
have been implanted with a gene that simulates the
synthesis of blue pigment in pansies.

Its scientists successfully pioneered implanting into

the flowers the gene that produces Delphinidin, the
primary plant pigment that produces a blue hue but
is not found naturally in roses. The Blue Rose
The world's first genetically modified blue roses was developed by
were unveiled in the laboratory four years ago, Suntory Flowers
although further research was required to make
them safe to grow in nature.
Protein Engineering Is Used to Improve a
Detergent Enzyme

• Subtilisin: is a serine protease produced by bacteria. Due

to its broad specificity for proteins that commonly soil
clothing, this enzyme was developed for commercial use
in laundry detergents.
• The first detergents containing subtilisin suffered from a
serious drawback:
they could not be used with bleach, because bleach
inactivates the enzyme. Biochemical analysis determined
that loss of activity was due to the oxidation of a
methionine at position 222.
 Protein Engineering Is Used to Improve a
Detergent Enzyme cont.
• Scientists decided to see whether a variant of
subtilisin could be produced that was no longer
sensitive to bleach.
• Site-directed mutants were constructed in the gene
encoding subtilisin. The strategy was simply to
substitute, one at a time, each of the non-wildtype
amino acids at residue 222.
• The mutant genes were cloned into expression
vectors and the 19 different subtilisin derivatives
were expressed.
 Protein Engineering Is Used to Improve a
Detergent Enzyme cont.

• Biochemical analysis showed that the Alanine-

substituted enzyme, which was 53 percent as active as
wildtype subtilisin. This variant exhibited no
detectable bleach sensitivity, so detergents containing
this engineered subtilisin can now be used with bleach.
(This new variant of subtilisin is an example of a
second generation molecule)
• Protein engineers are currently at work on a third
generation molecule that exhibits decreased
temperature sensitivity so that it can be used in hot
water.
Engineering Plants to Overcome
Biotic &Abiotic Stresses

Prof. Dr. Reda Moghaieb

Department of Genetics
Faculty of Agriculture
Cairo University
 What is Plant Stress

• Plant stress is the adverse reaction of plants to

environmental conditions that are unfavorable to growth,
such as lack of sufficient nutrients, inadequate watering,
flooding, high or low temp., disease or insect infestation.
 Types of stresses

Biotic Abiotic
Caused by living Caused by nonliving factors,
organisms, such as water (drought, flooding),
fungi, bacteria, viruses, extreme temperature (chilling,
nematodes, insects, freezing, heat), salinity, heavy
mites, and animals. metals (ion toxicity), and
nutrients availability.
 Salinity
• Salinity in the arid and semi-arid regions of the world as well
as in irrigated lands is a serious threat to agriculture, affecting
plant growth and crop yields (Zahran, 1999; Duzan et al.,
2004).

• Cultivatable Land (21% of land surface)

Cultivated (47% of cultivatable land)
Salt affected (13% of cultivated land )
• Irrigated Land (16% of cultivated land)
Salt affected (50% of Irrigated land)
Salt-Affected soils in different continents of
the world

Area (million ha) Continent

409.3 Asia
240.5 Africa
147.3 Australia
129.2 South America
50.8 Europe
17.5 North America
 Salinity problems
Soil salinity existed long time before humans and agriculture but the
problem has been aggravated by agricultural practices such as
irrigation. Today 20% of the world’s cultivated land and nearly half of
all irrigated lands are affected by salinity.

Salinity especially affects third world countries, forcing their people

to migrate since the technology to combat it is extremely costly, and
requiring large expenditures of energy to reclaim land and
maintaining salt balances.

On the other hand, these countries have been crucial for

establishing germplasm bank collections, therefore, efforts have to
be directed towards tailoring crop plants to suit more saline
environment.
 Evolution of salt tolerance

Soil salinity almost always originates from previous exposure to

seawater. Although it is believed that for most of the Earth's
history, the salt level of the oceans was much lower than now, all
plant species that inhabit the seas, as well as a phylogenetically
diverse groups of land plants, are capable of growth and
reproduction at salinity levels near or above those found in the
seas.

This strongly supports the existence of a genetic basis for high-

salinity tolerance within both sea and land plants.

Plant Physiol. 135, 1718-1737.

7
 Salt Stress Effects on Plants

Primary
• Water deficit
• Ion disequilibrium

Secondary
• Reduced membrane function
• Reduced assimilate production
• Increase carbon requirements
• Decrease cytosolic metabolism
• Reduced cell expansion
• Production of reactive oxygen
intermediates (ROIs)
• Programmed cell death
 What can plants do when confronted with environmental
stress?

Die
Flower and fruit rapidly
Adapt
Some physiological changes, generally interacted as adaptive, have
been shown to occur in salt-stressed plants. These include alterations in
ion transport, lipid composition and accumulation of organic osmolites.

Additional or other metabolic pathways：

• Overproduction of mannitol
• Trehalose production
• Synthesis of proline, glycinebetaine or other osmoprotectants
Salt Tolerance Classification of Plants

Relative Dry Weight Gain (%) 125 Euhalophytes

100

Miohalophytes
75

50

25
Glycophytes
0
0 100 200 300 400 500 600 700
[Cl-ext] (mM)
Adapted from Greenway and Munns. (1980) Annu Rev Plant Physiol. 31:149-190
Glycophytes vs halophytes - sweet plants and salt plants, respectively, by
definition halophytes are “native flora to a saline environment” Quantitative
difference – adaptation

Nearly all salt tolerant plants are angiosperms, indicating polyphyletic origin,
or halophytes are primitive genetic remnants of different families

Salt tolerant species exist in 1/3 of the angiosperm families; however about ½
of the 500 halophytic species belong to 20 families, monocots - 45 genera in the
Poaceae family and dicots - 44% of the halophytic genera are in the
Chenopodiaceae (Atriplex, Salicornia and Suaeda)

Most plants, including the majority of crop species, are glycophytes and cannot
tolerate high salinity.

11
Salt tolerance research is important basic
plant biology

• Salt tolerance research contributes to our understanding of

subjects ranging from gene regulation and signal transduction
to ion transport, osmoregulation and mineral nutrition.

• Additionally, some aspects of salt stress responses are intimately

related to drought and cold stress responses.
• Plant salt tolerance studies thus contribute to understanding
cross-tolerance

12
 Functional Proteins Structural Proteins
Membrane Proteins: Water Transcription factors: bZIP,
channel, transporter MYB, MYC, ERF/AP2

Proteinase:
chloroplast, cytoplasm
Protein Kinases:
MAPK, MPKK,
Protection factor of Drought CDPK
macromolecules:
chaperon, LEA Stress
Protein Phosphatases
Osmoprotectant (PTP)
synthesis: proline, Gly
betaine, sugar

Detoxification enzymes: PI turnover

GST, SOD (phospholipase C)
The three aspects of salt tolerance in plants (homeostasis,
detoxification and growth control), Zhu, 2001.
The three aspects of salt tolerance in plants (homeostasis,
detoxification and growth control) and the pathways that
interconnect them; homeostasis is broken down into ionic and
osmotic homeostasis. The SOS pathway mediates ionic homeostasis
and Na+ tolerance. A mitogen activated-protein kinase (MAPK)
cascade similar to the yeast HOG1 pathways is proposed to mediate
osmotic homeostasis. The two primary stresses, ionic and osmotic
stresses, cause damage or secondary stresses such as oxidation.
Lea type stress proteins such as RD29A are proposed to function in
the detoxification or alleviation of damages. CBF/DREB transcription
factors mediate some of the stress protein gene expression in
response to secondary stresses caused by high salt concentrations,
cold, drought or abscisic acid (ABA). The ionic homeostasis, osmotic
homeostasis and detoxification pathways are proposed to feed
actively into cell division and expansion regulation to control plant
growth.
Molecular scheme of abiotic stress signal transduction
pathway in plants

Signal
Perception

Signal
Transduction

Signal
Response
CURRENT SCIENCE, VOL. 88, NO. 11, 10 JUNE 2005
 Stress signal transduction
The signal transduction networks for cold, drought, and salt stress can be divided into
three major signaling types:

(I) Osmotic/oxidative stress signaling that makes use of MAPK modules; (the
production of compatible osmolytes and antioxidants, and may also relate to cell
cycle regulation under osmotic stress).

(II) Ca2+-dependent signaling that lead to the activation of LEA-type genes

(such as the DRE/CRT class of genes), and

(III) Ca2+-dependent SOS signaling that regulates ion homeostasis; (specific for
the ionic aspect of salt stress. Targets of this type of signaling are ion
transporters that control ion homeostasis under salt stress).
The SOS pathway functions in ion
homeostasis under salt stress
High extracellular concentrations
of salt elicit a rise in cytosolic
Ca2+. The Ca2+ sensor SOS3 upon
the perception of this signal
interacts with and activates the
protein kinase SOS2. Activated
SOS2 then regulates the ion
transporter activities or TFs to
regulate ion homeostasis or gene
expression. The SOS2 targets
include the SOS1 Na+/H+
antiporter, the vacuolar
Na+/H+ exchangers NHX,and the
Na+/K+ transporter HKT1. Other
targets include tonoplast ATPase
and pyrophosphtases,water
channels and K+ transporter
18
 Salt stress-regulating genes
SOS3 is a Ca2+BP that contains EF-hands and a myristoylation site in the N
terminus. It has homology with yeast calcineurin subunit B and animal Ca2+ sensors.

SOS2 is a Ser/Thr kinase similar to yeast sucrose nonfermenting (SNF1) kinase and
the mammalian cAMP-activated PK
SOS1 is a plasma membrane Na+/H+ antiporter resembling the mammalian NHE and
bacterial NhaP exchangers

SOS1 expression is upregulated by salt stress in plants but this upregulation is reduced
by sos3 or sos2 mutations

Salt stress elicits rapid increase in free cytoplasmic Ca2+. SOS3, a myristoylated
Ca2+BP, that can sense this calcium signal. SOS3 also recruits SOS2 to the plasma
membrane, where the SOS3-SOS2 protein kinase complex phosphorylates SOS1 to
stimulate its Na+/H+ antiport activity. Loss-of-function mutations in SOS3, SOS2, or
SOS1 cause hypersensitivity to Na+
 Studying the Salt stress
• 1) Physiology of salt toxicity and salt tolerance. This includes
cellular and metabolic responses to salt, as well as whole plant
responses.

• 2) Mechanisms of salt transport across cellular membranes and

over long distances. This includes physiological and molecular
characterization of ion transporters involved in salt uptake,
extrusion, compartmentalization

• 3) Survey genes whose expression is regulated by salt stress.

This research is accelerated by using microarrays

• 4) Mutational analysis of salt tolerance determinants and salt

stress signaling
20
 Different approaches to identify
abiotic stress-related genes in plants

1. QTL mapping

2. Molecular biological studies

ex: substractive hybridization, differential display, microarray etc.

3. Molecular genetic studies

RD29A promoter-Luciferase transgenic
Arabidopsis thaliana

4. Functional genomics studies

overexpression (transgenic approaches)
T-DNA or Tos17 mutants
(gain-of or loss-of function)
 Functional Genomics of Plant Stress Tolerance

• Complexity and Multigenicity of Stress Responses.

• 1. Variations on common physiological Themes.
• 2. Evolutionary Conservation of Stress Responses
Mutants with altered sensitivity to osmotic/salt stress
Mutants in stress signal transduction pathways using osmotically
regulated promoter-reporter screening
Identify Suppressors of Stress-responsive mutants

22
 Salt and drought stress ⚫
➢ Salt tolerance are often equivalent to drought tolerance
➢ Various proteins or compounds can be expressed
• Osmoprotectants (osmolytes)- sugars, alcohols, proline, quatenary
ammonium compounds, etc. (such as trehalose, proline, D-ononitol,
mannitol, sorbitol, glycine betaine, 3-dimethylsulfoniopropionate, poly
amine).
Function
1. Facilitate both water uptake and retention
2. Protect and stabilize cellular macromolecules from damage by high salt

• Plant stress proteins (e.g. chaperones, heat shock proteins)

• Reactive-oxygen-scavenging proteins (e.g. superoxide dismutase)
• Hormone biosynthesis and catabolism protein
(e.g. affect level of ABA, cytokinin, ethylene, etc.)
• Transcription factors or signaling proteins
 Trehalose
➢ A natural alpha-linked
dissacharide

Figure 19.31

➢ Use ABA-inducible promoter; fuse two enzymes into one.

➢ In the presence of salt, biomass is 4~6 X compared to the non-
transformed one in the presence of salt

Figure 19.32

24
 Delay the onset of drought-induced senescence

➢ itp gene: from Ti plasmid

➢ PSARK: senescence-associated
protein kinase promoter
➢ Require only 30% of water
➢ Produce 4~5X higher level of
biomass

25
⚫ Insect resistance
Why need insect-resistant plants?
➢ Cost down
➢ Specifically eliminate a limited number of insect species
➢ Non-hazardous to human or other higher animals
➢ Decrease other disease problems simultaneously

Genes resources
➢ Protoxin from Bacillus thuringiensis ，NOT Bacillus
subtilis
➢ -amylase inhibitors, protease inhibitors, lectin, etc. from
plants
 About Bt toxin

• Spores and crystalline insecticidal proteins produced by B.

thuringiensis have been used to control insect pests since the
1920s.
• They are now used as specific insecticides under trade
names such as Dipel and Thuricide.
• Because of their specificity, these pesticides are regarded as
environmentally friendly, with little or no effect on humans,
wildlife, pollinators, and most other beneficial insects.
 About Bt toxin

• B. thuringiensis-based insecticides are often applied as

liquid sprays on crop plants, where the insecticide must be
ingested to be effective.
• It is thought that the solubilized toxins form pores in the
midgut epithelium of susceptible larvae.
• Recent research has suggested that the midgut bacteria of
susceptible larvae are required for B. thuringiensis
insecticidal activity.
 Increasing expression of the Bt protoxin (Cry
protein)
➢ History of engineering Bt toxin expressions in transgenic
plant (Table 19.1)
• Low expressions of cry1Aa, cry1Ab, cry1Ac in the
beginning
• Use only insecticidal N-terminal domain (646 aa)
• Use strong promoter
• Change codons (PM, partially modified-increase 10X, FM,
fully modified-increase 100X)
• Target Bt protein into chloroplast (add transit peptide at
the N-termi), reach 1% expression level
• Chloroplast transformation (Fig. 19.2) via homologous
recombination, reach 2~3% expressions.
30
 Chloroplast transformation for Bt protoxin
expressions
➢ Advantages of homologous recombination? (so no
integration damage to other genes)
➢ Advantage of polycistronic arrangement? (no need to put
promoter for each single gene, but still require ribosome-
binding site for translation)
➢ Advantages of chloroplast transformation
1. No need to modify codons
2. High expression level as copy number increased
3. No risk of unwanted transfer of the protoxin genes
➢ Disadvantages of chloroplast transformation
Not expressed in non-green tissues such as fruits
Chloroplast genome

homologous
recombination
Construct

Figure 19.2

Both rbcL and accD are single copy gene in chl. genome •
Both ORF require its own ribosomal binding site (rbs) •
 Bt is not effective enough to all insects

➢ The extent of protection is not universal to all insects

➢ Combine to use low dose of chemical insecticide is better

➢ Gene stacking transgenic plant (gene pyramiding, box 19.2)

• ~300 toxin gene have been isolated from different strains

of B. thuringiensis
• Put two Bt genes in one crop (e.g. Cry1Ab2 and Cry 1Ac)
• Combine Bt gene and other insecticidal-toxin gene
 Other strategies for protecting plants against
insects-1
➢ Protease inhibitor
• Low expression level- 0.2% of total plant protein
• Not toxic to human
✓Common components in food
✓Generally degraded while cooking
✓Can use tissue-specific promoter
• How to increase the effectiveness?
✓Use together with low dose of Bt
➢ -amylase inhibitor
• Genes isolated from common bean (Phaseolus vulgaris)
• Use seed-specific promoter
• Inhibit the growth of seed feeding beetles
 Other strategies -2

➢ Cholesterol oxidase

Gene isolated from bacteria such as Streptomyces •

Catalyze the oxidation of 3-hydroxysteroids to ketosteroids and •
hydrogen peroxide.
Low expression is enough, 10 ppm= 0.001 % •
• Probably act by disrupting the insect’s midgut epithelial
membrane, thus killing the insect.
 Other strategies -3

➢ Vegetative insecticidal proteins (VIPs)

• Produced by B. thuringiensis during its vegetative growth
• Beside Cry proteins, more than 300 insecticidal toxic genes have
been identified from Bacillus
• Two major Vips
1. Vip1 and Vip2: not toxic to lepidoptera
2. Vip3: toxic to several major lepidoptera

• Use domain shuffling to increase its diversity

• Test its synergistic effect with Bt
 Other strategies -4

➢ Lectins (carbohydrate-binding proteins found in plants)

➢ Tryptophan decarboxylase
➢ Toxin A
➢ Avidin
✓ A glycoprotein from chicken egg
✓ May cause biotin deficiency by its high affinity at low dose (so
not toxic to animals)
 Other strategies -5

➢ Gossypol
• A yellow polyphenolic aldehyde, NOT a protein!!!
• Permeate cells and act as an inhibitor of several insect’s
dehydrogenase enzymes
• Produced by cotton naturally to prevent insect predation
• Can be inactivated by cytochrome P450 monooxygenase
Biosafety and Risk Assessment
of GM Plants
Prof. Reda Elwany Moghaieb
Professor of Molecular Genetics
Faculty of Agriculture, Cairo University
Egypt
 Food consisting of living organisms, e.g.
soybean, maize

Food derived from GMO e.g. soy oil, corn

flour

Foods containing ingredients produced by

GMO, e.g. Vitamins or essential amino acids

Foods containing ingredients processed by

enzymes produced by GMO, e.g. high
fructose corn syrup produced using
recombinant glucose isomerase
 Food from GM plant

Food

Characterization Toxicology Nutritional Eq… Q’litative C’tion

Subchronic rodent Broiler chicken

Comparison with dietary feeding (42 days) GM Food
non GM isogenic studies
parental line (rats, 90 days) As Safe As

Rapid growing sp
Compositional Biochemistry, Sensitive to
Haematology changed nutrition
Analyses
Histopathology
Agronomic Organ wt etc
characters
Yes No
Reference non GM
Tolerance limit
 Biosafety

• Biosafety is a set of actions focused on preventing,

minimizing and eliminating risks associated with
research, production, teaching, use, technology
development and services related to genetically
modified organisms (GMOs) with the aims of protecting
human and animal health and environmental
preservation.
 Food biosafety
•
The food safety of transgenic plants is assessed in
accordance with risk analysis.
• This methodology was initially developed with the aim of
assessing deleterious effects on human health arising from
potentially toxic chemicals present in food, pesticide
residues, contaminants and food additives and was
subsequently applied in assessing the food safety of GM
plants.
Biotech crop countries and mega-countries
The ten major producers of transgenic crops
in the world
 The risk assessments are performed in
three steps:

• Step 1. Risk assessment:

This step can be defined as the evaluation of the probability of
adverse health effects arising from human or animal exposure to a
hazard.

• Risk assessment consists of four segments:

i. Hazard identification, which entails the identification of

biological, chemical and physical hazards found in food that may
cause adverse health effects;
• ii. Hazard characterization, which entails an evaluation of
an identified hazard in qualitative and quantitative terms
and often involves the establishment of a dose response
relationship due to the magnitude of exposure (dose) to a
physical, chemical, or biological hazard and the severity of
adverse health effects;

iii. Exposure assessment, which entails a quantitative and

qualitative assessment of the likelihood of ingestion of
physical, chemical and biological agents through food;

iv. Risk characterization, which entails a qualitative and

quantitative estimation of the likelihood and severity of an
adverse effect on health based on identification and
hazard characterization and on exposure assessment.
Step 2. Risk management:

• Risk management is measured from the results

of risk assessment and other legitimate factors
to reduce risks to the health of consumers.
•
Measure of risk management may include
labeling, imposition of conditions for
marketing approval and post-trade monitoring.
Step 3. Risk communication:

• Risk communication includes the information exchange

that must occur between all stakeholders, including the
government, industry, the scientific community, media and
consumers.
• It should occur throughout the assessment and risk
management processes and should include an explanation
to the public of the decisions made, ensuring access to
documents obtained from the risk assessment and, at the
same time, respecting the right to safeguard the
confidentiality of industrial information…………………..
 Environmental biosafety

• Similar to food risk assessment, environmental risk

assessment considers three important points: the
possibility, probability and consequences of a hazard,
which should always be assessed on a case-by-case
basis.
• This means that, following the identification of a
possible danger, you should consider whether that
danger is possible, if it is likely and, if it were to
occur, what the result would be?.......................................
 Gene escape from transgenic plants can
occur in three main ways:

• i. When the transgenic plant becomes a weed or an

invasive species (e.g., for crops with weed-like
characteristics, such as sunflower, canola, and rice), the
transgenic gene found in the transgenic plant may allow
the crop to become weedier and more invasive;
•
ii. Intraspecific and interspecific hybridization, such as
when transgenic DNA is transferred by crossing to other
varieties of cultivated species and wild species,
respectively;
•
iii. When transgenic DNA is asexually transmitted to other
species and organisms.
 For a gene to escape and be transferred to
different species, certain conditions are
necessary:

• i. The two parental individuals must be sexually compatible.

• ii. They must be located in neighboring areas and with
flowering overlap between the two parental types.
• iii. A sufficient amount of viable pollen must be present and
transferred between individuals.

• iv. The resulting progeny should be fertile and ecologically

adapted to environmental conditions where the parents
are located.
• To avert gene escape from transgenic varieties to
conventional varieties, isolation distance should be
maintained. For example, maize is a wind-pollinated
species, and the distances that pollen can travel depend on
the wind pattern, humidity and temperature. In general,
fields with transgenic varieties should be isolated from
other conventional varieties with a distance of at least 200
m
 Approaches to risk assessment

1. Trait analysis
– characteristics of the modified organism; transgene,
parental organisms, receiving environment
– less problem, if small scale
– more problem, if large scale

2. Familarity
– comparison of transgenic to similar organism(s) derived
from classical genetic methods
– assume that small genetic changes (1-4 genes) exhibits no
significant change in well-known organism, phenotype is
still the same
3. Formulaic
– possible adverse effects; to human health or the
environment
– R=HxE
– R; Risk, H; Hazard, E; Exposure
– facilitates consideration of risk-management
options

4. Intuitive Reasoning
– use education, experience and reason to promote
knowledge for making decision with complete
information
– depends on what should be considered
– use of expert committees, independent
reviewers/assessors without a conflict of interest
Guidelines for Plant Testing
 1. Field obseravation
Emergence Order of testing relies on degree of
possible risk of the plant

Growth Transgenic plant growth / wild type,

measurement not greater than 1
Days of flowering Transgenic plant days of flowering /
wild type, not greater than 1

Length of Transgenic plant length of flowering

flowering period period / wild type, not greater than 1

Pollen dispersal This is the reason why buffer zone

distance has to be set up)
Shattering of seed Distance of seed shattering
from plant determines degree of risk
Reproductive success Reproductive success
or yield (annual) determines transmission risk
Reproductive success Perennial risk determines more
or yield (perennial) risk
Qualitative insect Non-target insects = 0

Qualitative Non-target pathogens = 0

pathogens
Others
 2. Plant testing
Dormacy/ germination -shorter dormancy / germination
determine front running risk
-longer dormancy / germination
determine latent risk
risk -increase longevity determines risk
Field seedbank longevity
(dormancy x viability)

Competition -stronger competition determines risk

(Replacement or
addition series)
Replacement capacity -higher replacement capacity
determines risk
Gene flow (through -wider pollen dispersal determines risk
pollen movement) -outcrossing determines risk

Introgression -hybrid weediness determines long term

(hybrid weediness) risk

Alleopathy -Competitive of survival risk

Susceptibility to -Competitive of agricultural risk
conventional
management
Genetic stability -High genetic stability determines risk
Epistasis -Epistasis determines unexpected genetics
-Horizontal gene transfer
◼ Gene transfer between plant nucleus and

organelle
◼ Gene transfer between plant nucleus and

genome of consumer, predator, Gene

transfer between nucleus and organelle

Other
 Regulatory principles:

1. Scientifically based, based on information of organism,

used technology and effects to humans and environment
2. Product-based approach, use existing product-based
legislation
3. Familiarity and substantial equivalence, experience with the
use of that species. The determination is based on scientific
literature and practical experience with the plant and
similar plant varieties.
4. Case-by case, allow the development of knowledge that
could inform criteria and requirement over time.
 Regulatory principles:

5. Step-wise fashion, products should be assessed throughout

the chain of development : From laboratory to greenhouse
and finally large-scale field trial
6. Transparency
7. Precautionary principle/approach, derived from Rio
Declaration, regulatory groups can make decisions about
products based on scientific uncertainty.
8. Harmonization, sharing of or acceptance of another group’s
review
Risk management of transgenic
plants
1. Good laboratory practice

• Tightly control of GM-vectors, plasmids and plant

materials
• Apply no bacterial antibiotic resistant-derived gene
• Apply bioluminescence gene from animal as marker
• Apply antisense for pollen developmental gene
• Limit level of toxic gene, eg, cry family
2. Good agricultural practice

• Controlled plantation area with standard buffer zone

and % sharing with wild type plants
• Emasculation
• Flower bud elimination
• Closed-bag control
• Net protection of fruits and seeds from insects, birds,
bats, rodents
• Total fruit and seed collection
• Labeling and separation technique for transgenic
plant and seed
• Whole plant elimination after harvest
3. Good manufacturing practice

• Labeling GM-products according to domestic and export

regulations
• Testing for allergen and toxicity of the products
containing GM-materials
 4. Good marketing practice

• Fully-informed alien gene(s) and awareness of

application
• Evaluated for allergen and toxic molecule
• Labeling
• Post marketing record
 5. Good consumption practice

• For GM-food products: determine animals as primary

consumer and human as secondary consumer
• Study labeling
• Food safety criteria
The Role of Biotechnology In Food Security

Prof. Reda Elwany Moghaieb

Professor of Molecular Genetics
Faculty of Agriculture, Cairo University
Egypt
 “The greatest challenge of the 21st century:
feeding 9 billion people with a sustainable
agricultural production system.”

To meet these challenges in the 21st Century and beyond,

humanity will need ever tool it can find.
 Dietary Problems

Overnourished

Undernourished People who suffer from

overnutrition, receive
too many calories each
day, live largely in the
Undernourished people developed world
receive less than 90% of
their daily caloric needs
and live mostly in
developing countries.
 Dietary Problems

Malnutrition, the lack of

nutritional elements the body
needs, including a complete
complement of vitamins and
minerals, can occur in both
undernourished and
overnourished individuals.
 Agriculture production and population increase

Our ability to produce Picture 9.1

food has grown even
faster than has global
population.

Due to political obstacles

and inefficiencies in
distribution, almost
800 million people in
developing countries
do not have enough to
eat.
We have genetically modified food for thousands
of years

• The earliest farmers and gardeners saved seeds of the very best
plants to start the next growing season
• By doing this, they unknowingly selected plants with the more
desirable genes

Assyrian mural from 870 BC

showing palm pollination
 4,500
Years Ago

Prickly lettuce

9000
Years Ago

Corn
Some crops never existed in
nature
• Wheat, Triticum aestivum
Triticum urartu X Aegilops speltoides
2n=14 2n=14

Triticum turgidum X Aegilops tauschii

2n=28 2n=14

Triticum aestivum
2n=42
 • Increases Crop yield
• Drought tolerant crops
Agricultural • Pests resistant Crops
Industry • Disease resistant Crops

• Biofuel production
• Reducing waste
Renewable
• Pollution control
Energy

• Development of vaccine
• Hormones e.g. Insulin
Pharmaceutical • Gene thereby
Industry • Engineered enzymes
Most Frequent Categories

Source: U.S. Department of Agriculture

Tomatoes comes in many varieties,
.colors and shapes.Transgenic tomatoes -
expressing different malarial antigens
 Example: Rennin Production

 The protein rennin is used to

coagulate milk in the
production of cheese.
 Rennin has traditionally been
made in the stomachs of calves
which is a costly process.
 Now scientists can insert a
copy of the rennin gene into
bacteria and then use bacterial
cultures to mass produce Rennin in the top test tube… not there in
rennin. the bottom one.
 This saves time, money, space
and animals.
Extended Shelf Life

Extended Shelf Life Milk

 Better Nutrient Composition

 Some plants, during processing,

lose some of the vital nutrients they
once possessed.
 Others are grown in nutrient poor
areas.
 Both these problems can be solved
by introducing genes into plants to
increase the amount or potency of
nutrients.
 “Biofortification”
 Transgenic Fish
Antifreeze Proteins (AFP)
Tilapia
AFPs lower the freezing Salmon/trout
temperature of blood & fluids
Catfish
Trout normally do not survive in
Transgenic
water below –0.6°C Can grow up to 6 times faster than
Wildtype wildtype fish
Transgenic trout containing an
AFP gene & promoter can survive Most have extra copies of growth
in waters as cold as –1.2°C hormone (GH) gene
 Industrial uses
• Cleaning industry
– Detergent proteases
• Textile industry
– Finishing cloth
– Better cotton fibers
• Paper and pulp industry
– Processing with biotech, environmentally friendly
chemicals
Contribution of biotech crops to food security,
sustainability, and climate changes