GENETIC Kabanda Stephen

TECHNOLOGY
Principles Of
Genetic
Technology
▪ Genetic engineering: this is the deliberate manipulation
of genetic material to modify specific characteristics of
an organism
▪ This may involve transferring a gene into an organism so
that the gene is expressed
▪ The organism is then said to be a genetically modified
organism (GMO)
▪ Recombinant DNA (rDNA): DNA made by artificially
joining together pieces of DNA from two or more
different species
▪ Transgenic organism: any organism that contains
DNA from another source, such as from another
individual of the same species or from a different
species
▪ Genetically modified organism (GMO): any
organism that has had its DNA changed in a way
that does not occur naturally or by selective
breeding
▪ Vector: a means of delivering genes into a cell
used in gene technology; e.g., plasmids and
viruses
▪ Promoter – a length of DNA (usually about 40 bases
long) situated next to genes and which includes the
binding site for RNA polymerase where transcription of
a gene or genes begins
▪ In eukaryotes, promoters also have sites for binding of
transcription factors
▪ Marker – a gene which is deliberately transferred along
with the required gene during the process of genetic
engineering
▪ It is easily recognised and used to identify those cells to
which the gene has been successfully transferred
▪ They code for identifiable substances that can be tracked
(e.g. GFP - green fluorescent protein which fluoresces
under UV light)
▪ Genetic fingerprinting – the analysis of DNA in
order to identify the individual from which the
DNA was taken to establish the genetic
relatedness of individuals
▪ It is now commonly used in forensic science (for
example to identify someone from a blood
sample) and to determine whether individuals of
endangered species in captivity have been bred or
captured from the wild

▪ DNA sequencing - the determination of the

precise sequence of nucleotides in a sample of
DNA or even a whole genome e.g., the Human
Genome Project
▪ Gene editing: a form of genetic engineering in
which the genome of an organism can be changed
by deleting, inserting or replacing a length of
DNA at specific sites in the genome using a
method such as the Crispr/Cas9 system
Genes to be transferred into an organism
may be: Extracted from the DNA of a
donor organism

▪ The extraction of the gene (containing the desired

nucleotide sequence) from the donor organism occurs
using restriction endonucleases
▪ Restriction endonucleases are a class of enzymes from
bacteria which recognise and break down the DNA of
viruses that infect bacteria known as bacteriophages
▪ These enzymes cut the sugar–phosphate backbone of
DNA at specific places within the molecule
 Sticky Ends
▪ Restriction enzymes either cut straight
across the sugar–phosphate backbone to
give blunt ends or they cut in a staggered
fashion to give ‘sticky ends’
▪ Sticky ends are short lengths of unpaired
bases
▪ They are known as sticky ends because
they can easily form hydrogen bonds with
complementary sequences of bases on
other pieces of DNA cut with the same
restriction enzyme The restriction enzyme, BamHI, makes staggered
cuts in DNA to give sticky ends
Genes to be transferred into an organism
may be: Synthesised from the mRNA of
a donor organism by reverse
transcription

▪ The isolated mRNA is combined with a reverse

transcriptase enzyme and nucleotides to create a
single strand of complementary DNA (cDNA)
▪ DNA polymerase is then used to convert the single
strand of cDNA into a double stranded DNA molecule
which contains the desired code for the gene
▪ This technique for isolating the desired gene is
considered advantageous as it forms mRNA with only
sequences of bases (called exons) that code for
sequences of amino acids and no introns
Genes to be transferred into an organism
may be: Synthesised from the mRNA of
a donor organism by reverse
transcription
Genes to be transferred into an organism
may be: Synthesised chemically from
nucleotides
▪ The sequence of nucleotides is held in a computer
that directs the synthesis of short fragments of DNA in
DNA synthesiser machines
▪ These fragments are then joined together to make a
longer sequence of nucleotides that can be inserted
into plasmids for use in genetic engineering
▪ This method is used to generate completely new
genes that are used, for example, in the synthesis of
vaccines
Genes to be transferred into an organism
may be: Synthesised chemically from
nucleotides
The following are needed to
produce a GMO:
▪ Enzymes, such as restriction endonucleases, DNA
ligases, DNA polymerase and reverse transcriptase
▪ Vectors, including plasmids and viruses
▪ Genes coding for easily identifiable substances that
can be used as markers
 Enzymes used in gene technology:

Enzyme Natural source Application in genetic

engineering
Restriction enzyme cytoplasm of bacteria (combats breaks DNA molecules into shorter
(restriction endonuclease) viral infection by breaking up viral lengths, at specific nucleotide
DNA) sequences
DNA ligase with nucleic acid in the nucleus of joins together DNA molecules
all organisms during replication of DNA
DNA polymerase with nucleic acid in the nucleus of synthesises nucleic acid strands,
all organisms guided by a template strand of
nucleic acid
Reverse transcriptase in retroviruses only synthesises a DNA strand (cDNA)
complementary to an existing RNA
strand
 Plasmids
▪ These are small, circular pieces of
double-stranded DNA. Plasmids occur
naturally in bacteria and often contain
genes for antibiotic resistance.
▪ They can be exchanged between bacteria
– even between different species of
bacteria. If a genetic engineer inserts a
piece of DNA into a plasmid, then the
plasmid can be used to take the DNA into
a bacterial cell
 Properties Of
Plasmids That Allow ▪ They have a low molecular mass, so they are readily
Them To Be Used In taken up by bacteria
Gene Cloning ▪ They have an origin of replication so they can be
copied/can replicate independently and fast
▪ They have several single target sites for different
restriction enzymes in a short length of DNA called a
polylinker
▪ They have one or more marker genes, allowing
identification of cells that have taken up the plasmid
 Properties Of
Plasmids That Allow
▪ They are circular double stranded DNA so, more stable
Them To Be Used In or not damaged by host cell enzymes
Gene Cloning: ▪ They can be cleaved by restriction enzymes, leaving
contd….. sticky ends
▪ May contain antibiotic resistance genes which can be
used as marker genes
▪ They can be easily extracted from bacteria
▪ May carry a promoter so gene can be expressed /
transcribed
 Why Promoters Need
To Be Transferred With
Desired Genes When
Producing A Genetically ▪ The expression of genes, such as those in the lac
Modified Cell operon, is controlled by a promoter
▪ This is the region of DNA to which RNA polymerase
binds as it starts transcription
▪ The promoter, initiates transcription/switches on gene
as it facilitates the binding of, RNA polymerase or
transcription factors
▪ Otherwise, the gene must be inserted near an existing
promoter. However, this is difficult to do, or it may
disrupt expression of existing gene
 Why Promoters Need To Be
Transferred With Desired Genes When
Producing A Genetically Modified Cell
▪ When bacteria were first transformed to produce
insulin, the insulin gene was inserted next to the β-
galactosidase gene so that they shared the same
promoter
▪ The promoter switched on the insulin gene when the
bacterium needed to metabolise lactose
▪ So, when bacteria were grown in a medium
containing lactose but no glucose, they synthesised
both β-galactosidase and human insulin
▪ In eukaryotes, proteins known as transcription
factors are also required to bind to the promoter
region or to RNA polymerase before transcription
can begin
 Process of how the
gene coding for human
insulin can be obtained
and inserted into a
plasmid vector
Inserting a human
gene into the
plasmid pBR322
 Process of how the
gene coding for human
insulin can be obtained ▪ Obtain mRNA with the code for human insulin from β
and inserted into a cells (of islets of Langerhans of pancreas)
plasmid vector ▪ The mRNA is incubated with a mixture of free DNA
nucleotides and the enzyme reverse transcriptase
▪ This produces a single strand of DNA known as
complementary DNA or cDNA
▪ The single strand of cDNA was then made double
stranded using the enzyme DNA polymerase, and
cloned to make many cDNA molecules using the
polymerase chain reaction (PCR)
▪ A restriction enzyme is then used to create sticky ends
▪ Plasmids are obtained from bacterial cells
Getting the plasmids ▪ Plasmids from bacteria are cut with the same
into bacteria restriction enzyme to produce complementary sticky
ends

▪ cDNA (insulin gene) and plasmids are mixed with the

enzyme DNA ligase
▪ DNA ligase seals the sugar phosphate back bone to
form recombinant DNA
Getting the plasmids
into bacteria ▪ First, the bacteria and plasmids are put into a solution
with a high concentration of calcium ions, then the
mixture is cooled and given a heat shock
▪ This increases the chances of plasmids passing
through the cell surface membrane of the bacteria. A
small proportion of the bacteria, perhaps 1%, take up
plasmids with the gene and are said to be
transformed
▪ The rest either take up plasmids that have closed
without incorporating a gene or do not take up any
plasmids at all
 Getting the
plasmids into
bacteria
 The Use Of Genes For Fluorescent Or Easily Stained Substances As
Markers In Gene Technology

▪ Due to risks of using genes for antibiotic resistance as

markers, genes for enzymes that produce fluorescent
substances are used.
▪ E.g., genes for enzymes from jelly fish which make a
protein called GFP (green fluorescent protein) that
fluoresces bright green in ultraviolet light
▪ The marker gene is placed in the plasmid close to where
the gene of interest is inserted
▪ It is easy to identify the transformed bacteria as the
transgenic organisms will glow green under u.v light
▪ Another marker is the enzyme β-glucuronidase (known as
GUS for short), which originates from E. coli
 Gene editing
This is a form of genetic engineering
in which the genome of an organism
can be changed by deleting, inserting
or replacing a length of DNA using a
method such as the Crispr/Cas9
system
Gene editing
▪ Until recently, genetic technology relied on relatively
poor methods of editing DNA
▪ For example, a modified virus could be used to insert
DNA into the human genome, but genetic engineers
had no control over where in a cell’s genome the DNA
is inserted
▪ It might be inserted in the middle of another gene,
with unpredictable consequences
Gene editing
▪ A technique called Crispr/Cas9 has changed that.
Crispr/Cas9 has been developed from a mechanism
used by some bacteria to defend themselves against
bacteriophages
▪ Crispr (pronounced ‘Crisper’) is a group of base
sequences that code for short lengths of RNA that
direct a nuclease enzyme known as Cas9 towards
specific base sequences
▪ The Crispr-associated (Cas) enzyme is an
endonuclease that cuts DNA at a point that is
determined by these RNA molecules, known as guide
RNA (gRNA)
Gene editing
▪ Part of each gRNA has a sequence of 20 bases that
can locate and bind to a strand of DNA with the
complementary base sequence. The enzyme Cas9 has
two active sites to cut DNA across both strands
▪ The Crispr/Cas9 system has been developed so that
gRNA can be made with base sequences that are
complementary to any base sequence of DNA
Gene editing

▪ After the DNA is cut by Cas9,

natural DNA repair mechanisms
can repair the break by:
▪ adding one or more
nucleotides that change the
base sequence
▪ inserting a short length of
prepared double-stranded
DNA with a specific base
sequence
Gene editing
▪ Crispr/Cas9 technology is used to:
▪ remove DNA, for example removing a ‘faulty’
allele or removing part of the faulty allele so that,
instead of producing a misfunctioning protein, it
does not code for anything that functions
▪ replace a faulty allele with a functioning one or
replace part of a faulty allele so that it then codes
for the functioning protein
 The
Polymerase
Chain Reaction
(PCR)
An automated process that amplifies
selected regions of DNA using
alternate stages of polynucleotide
separation (denaturation of DNA)
and DNA synthesis catalysed by DNA
polymerase
 The
polymerase
chain reaction

Note: ‘Primers’ are short

sequences of single-stranded
DNA made syntheically with
base sequences complimentary to
one end (the 3‘ end) of DNA

Remember: DNA polymerase

synthesises a DNA strand in the
3’ to 5’ direction
The polymerase chain reaction
▪ This is a method used for rapid production of a large
number of copies of a particular fragment of DNA
▪ First, the DNA is denatured, usually by heating it.
This separates the DNA molecule into its two strands,
leaving bases exposed
▪ The enzyme DNA polymerase is then used to build
new strands of DNA against the exposed ones
▪ A primer is used to begin the process. The primer
attaches to the start of the DNA strand, and then the
DNA polymerase continues to add nucleotides all
along the rest of the DNA strand
The polymerase chain reaction
▪ The process is repeated over several times each stage
at a different temperature
▪ The three stages in each round of copying need
different temperatures
 Stage 1
(denaturation):

▪ Denaturing the double-stranded DNA

molecules to make single-stranded
ones requires a high temperature,
around 95 °C
▪ This breaks the hydrogen bonds
between base pairs and separates the
double-stranded DNA molecules into
their two strands, leaving bases
exposed
 Stage 2 (annealing):
▪ The primers bind to the base sequences
on either side of the length of DNA which
is being amplified. They do this by
forming hydrogen bonds
▪ Primers are required because DNA
polymerase cannot begin synthesising
DNA without an existing strand on which
to build
▪ Attaching the primers requires a
temperature of about 60 °C
Stage 3 (extension):

▪ Building up complete new DNA

strands using DNA polymerase
(known as elongation) requires a
temperature of around 72 °C
▪ The DNA polymerases (Taq
polymerase) used for this process
come from microorganisms that
have evolved to live in hot
environments
 ❑Taq polymerase was the first heat-stable DNA
polymerase to be used in PCR. It was isolated from
thermophilic bacterium, Thermus aquaticus, which is
found in hot springs in Yellowstone Park in the USA

Taq ❑It is valuable for PCR for two reasons:

▪ It is not destroyed by the denaturation step, so it does
polymerase not have to be replaced during each cycle
▪ Second, its high optimum temperature means that the
temperature for the elongation step does not have to
be dropped below that of the annealing process. The
process is more efficient or faster than normal DNA
polymerase
Gel
Electrophoresis
 Electrphoresis

▪ The gel was placed in the tank containing a

suitable buffer solution
▪ Protein samples stained red have been added to
wells along the top of the gel
▪ They are migrating downwards towards the
anode
Electrophoresis
▪ A region of DNA that is known to vary between different
people is chosen. These regions often contain variable
numbers of repeated DNA sequences and are known as
variable number tandem repeats (VNTRs)
▪ Only identical twins share all their VNTR sequences
▪ The quantity of DNA increased by PCR. The DNA is then
fragmented by, restriction enzyme(s) / endonuclease(s)
▪ It is then loaded into wells in agarose gel at the cathode in
a buffer solution
Electrophoresis
▪ Direct current is applied, the phosphate groups of
DNA have a negative charge and are thus DNA
attracted to, the anode
▪ Short pieces/smaller mass move further/move faster
▪ The pieces are then transferred to a nylon membrane
and heated to separate the strands
▪ Probes or a fluorescent dye is applied and then
exposed to an X-ray film
▪ A pattern of stripes is then observed on the X-ray film
An electrophoresis
tank containing a gel
with DNA samples in
the wells. The tank
is filled with a buffer
solution and is
connected to the
power supply. The
samples of DNA will
move towards the
anode (positive
electrode)
Using DNA
profiling in
paternity testing
Using DNA
profiling in crime
scene analysis
Using DNA
profiling in crime
scene analysis
Electrophoresis
❑The movement of charged molecules within the gel in
response to the electric field depends on a number of
factors. The most important are:
▪ net (overall) charge – negatively charged
molecules, such as DNA, move towards the anode
(+) and positively charged molecules move
towards the cathode (–); highly charged molecules
move faster than those with less overall charge
▪ size – smaller molecules move through the gel
faster than larger molecules
▪ composition of the gel – the size of the ‘pores’
within the gel determines the speed with which
fragments of DNA move
 MICROARRAYS
▪ They are used to identify the genes present in an
organism’s genome and to find out which genes are
expressed within cells
▪ They allow the study very large numbers of genes in a
short period of time
▪ A microarray is based on a small piece of glass or
plastic usually 2 cm2
▪ Microarrays can be used to compare the genes
present in two different species
 ❑Populations of European ash trees, Fraxinus excelsior, are
susceptible to a chronic tree disease called ash dieback.
Since 2012, this disease has spread through Europe
causing large-scale loss of woodland
How microarray analysis
can be used to identify ▪ Ash dieback is caused by a fungal pathogen
the genes switched on
in an organism. ▪ Symptoms include stem lesions, death of growing
Example: shoots and wilting of leaves

▪ To limit the spread of the disease, 693 hectares of ash

woodland were cleared in the UK between 2012 and
2015 Clearing the woodland involved uprooting the
trees and burying them
 ▪ It was noticed that, in all woodlands infected with ash
dieback, there were some trees that were not affected

How microarray analysis ▪ Investigations were carried out across Europe to identify
the genes responsible for tolerance to ash dieback, using
can be used to identify a range of techniques including microarrays
the genes switched on
in an organism. ▪ Using the results of this research scientists hope to be
Example: able to replant forests with ash trees tolerant to ash
dieback

▪ Microarrays are used to detect the expression of

thousands of genes at the same time
 ▪ A slide is printed with thousands of spots in defined
positions
▪ Each spot contains single-stranded DNA of known
sequence, which acts as a probe to detect gene
expression
▪ mRNA samples are collected from healthy ash trees and
Stages of microarray from ash trees showing symptoms of ash dieback
analysis: ▪ Each sample of mRNA is converted to complementary
DNA (cDNA) using reverse transcription and fluorescently
labelled
▪ Different colour fluorescent dyes are used for the samples
taken from the healthy and diseased trees
▪ The two samples are added to the slide and allowed to
hybridise onto the DNA on the microarray slide (i.e., cDNA
hybridises to the probes / ssDNA on the microarray)
 Stages of microarray analysis:

▪ After hybridization, excess cDNA is washed off

▪ The microarray slide is then scanned to measure the
expression of each gene
▪ UV light or laser scanning is used to record fluorescence
pattern
▪ Intensity of fluorescence gives a quantitative measure of
gene expression
▪ A high intensity indicates that many mRNA molecules were
present in the sample, while a low intensity indicates that
there were very few
How to use a microarray to
compare the mRNA
molecules present in
cancerous and non-
cancerous cells. The results
identify which genes in the
cancerous cells that are not
normally expressed are
being transcribed
How to use a
microarray to
compare the mRNA
molecules
Genetic Technology
Applied
To Medicine
BIOINFORMATICS
This is the use of computer
programs or software to analyse
biological information from
databases
 ▪ Identify or recognise genes
The sequencing of ▪ Predict the primary structure of proteins
genomes and the use of
▪ Predict the tertiary/3D structure of proteins
bioinformatics can be
used to control ▪ Identify or predict the functions of a protein from the 3D
structure
parasites such as
▪ Drugs which can bind to the active site of the enzyme, or
Plasmodium through which can disrupt the structure of the protein are made
the following ways
▪ The drug may then prevent the transcription or
expression of a gene
 ▪ It is identical to human insulin
▪ There is a more rapid response
Advantages Of Treating ▪ There are no or fewer immune response, side effects or
Diabetic People With allergic reactions
Human Insulin ▪ It is cheaper to produce in large volume thus unlimited
Produced By Gene availability
Technology ▪ There is less risk of transmitting diseases
▪ It is good for people who have developed tolerance to
animal insulin
▪ There are no ethical, moral or religious, issues
 ▪ Genetically modified hamster cells are used to produce
factor VIII
▪ This protein is essential for blood clotting, and people
who cannot make it are said to have haemophilia
Factor VIII – a blood
▪ The human gene for making factor VIII has been inserted
clotting protein into hamster kidney and ovary cells which are then
cultured in fermenters
▪ The cells constantly produce factor VIII which is extracted
and purified before being used to treat people with
haemophilia
 ▪ Deficiency of the enzyme adenosine deaminase (ADA)
leads to accumulation of deoxyadenosine which is toxic to
T and B lymphocytes
Adenosine deaminase
for treating severe ▪ High yields of the enzyme ADA, which is used to treat
combined severe combined immunodeficiency disease (SCID), are
immunodeficiency made by a genetically modified insect larva, the cabbage
looper moth caterpillar
(SCID)
▪ This enzyme is administered to patients while they are
waiting for gene therapy or when gene therapy is not
possible
 Genetic
Screening
▪ Genetic screening is the
analysis of a person’s DNA to
check for the presence of a
particular allele

This can be done in adults,

in a fetus or embryo in the
uterus, or in a newly formed
embryo produced by in vitro
fertilisation
 How genetic screening is carried out
amniocentesis Chorionic villus sampling DNA profile

Tests foetus Tests foetus Tests possible carrier, parent

embryo
Sample amniotic fluid Sample chorionic villi of placenta Sample blood, skin, hair, cell from
IVF embryo
13-16 weeks pregnancy; 9-12 weeks pregnancy;
Results 2-3 weeks later; Results quicker than Results in hours;
Cells cultured to divide amniocentesis; DNA fragmented with restriction
Cells cultured to divide enzymes
Exploded into water; Exploded into water; Electrophoresis
Karyotyped Karyotyped
Fluorescent marker or gene Fluorescent marker or gene Radioactive gene probes;
probes; probes; Altered pattern of bands
Chromosomes examined and Chromosomes examined and Examined under UV light
counted counted
▪ In chorionic villus sampling, an ultrasound
scanner is used to guide the needle to the
placenta to remove a small sample of the fetal
chorionic villi which are embedded in the
placenta
▪ A small sample of the fetal blood is removed
for analysis
 Advantages of
screening for genetic
conditions
Genetic screening has a number of advantages;
▪ An adult woman with a family history of breast cancer
may choose to be screened for the faulty alleles of the
genes Brca-1 and Brca-2, which considerably increase an
individual’s chance of developing breast cancer.
▪ Should the results be positive, the woman may elect to
have her breasts removed (elective mastectomy) before
such cancer appears
 ▪ It provides information about the increased risk
Advantages of of person having genetic conditions e.g., breast
screening for genetic cancer in people with faulty alleles of BRCA2.
Should the results be positive, the woman may
conditions elect to have her breasts removed (elective
mastectomy) before such cancer appears
▪ It allows people to prepare for late onset genetic
conditions e.g., Huntington’s disease /
Alzheimer’s disease
▪ To identify whether fetuses are going to develop a
genetic condition, so can give early treatment
when born
▪ Allows parents to prepare for the birth of a child
who will need treatment for a considerable time
or even throughout life
 ▪ Identifies carriers of genetic conditions
▪ Helps to provide early diagnosis
Advantages ▪ It allows couples who are both carriers of a
of screening genetic condition to make decisions about
starting a family / having more children /
for genetic seeking IVF or even termination
conditions ▪ If negative, it removes anxiety
▪ Preventative treatment may be cheaper than
treating disease itself
 ▪ If positive, it could lead to social or financial
discrimination e.g., life insurance refusal
Disadvantages ▪ If positive, it may lead to anxiety, yet one may
of genetic still not develop the disease
screening ▪ If positive, it could lead to anxiety/worry
especially for illnesses that don’t have
treatment
 ▪ Give information about the severity of the
disorder or quality of life
▪ Give information about the available
treatments
▪ Give information about the components of diet
Role of a to avoid
Genetic
▪ Give information about the time of onset
Counsellor
▪ Explain the probability of the risk of passing on
the condition
▪ Explain the options available in light of religion,
culture or ethics
▪ Put individuals in a position to make their own
choices
 Gene Therapy
Gene therapy involves the cure of genetic
disorders by inserting ‘normal’ alleles of these
genes into the cells
 Gene Therapy

The most common vectors that

There have been problems in are used to carry the normal
getting normal alleles of the alleles into host cells are viruses
genes into a person’s cells and (often retroviruses or
then making them work properly lentiviruses) or small spheres of
when they get there phospholipid called liposomes.
Occasionally ‘naked’ DNA is used
Use of liposomes
 ▪ Cystic fibrosis is a genetic disorder in which abnormally
thick mucus is produced in the lungs and other parts of
the body
Cystic Fibrosis ▪ A person with cystic fibrosis is very prone to bacterial
infections in the lungs because it is difficult for the
mucus to be removed, allowing bacteria to breed in it
▪ Cystic fibrosis is caused by a recessive allele of the
gene that codes for a transporter protein called Cystic
Fibrosis Transmembrane Regulator (CFTR)
▪ This protein sits in the cell surface membranes of cells
in the alveoli, pancreatic duct, sperm duct and allows
chloride ions to pass out of the cells
▪ The recessive allele codes for a faulty version of this
protein that does not act properly as a chloride ion
transporter
 The CFTR (cystic fibrosis transmembrane conductance regulator)
protein in a plasma (cell surface) membrane

▪ The CFTR protein normally acts as a chloride channel where

Cl- move out of cells by active transport
▪ This results in a relatively high concentration of chloride
ions outside the cells. This reduces the water potential
below that of the cytoplasm of the cells
▪ So, water moves out of the cells by osmosis, down the
water potential gradient. It mixes with the mucus there,
making it thin enough for easy removal by the sweeping
movements of cilia
▪ However, in someone with cystic fibrosis, much less water
moves out of the cells, so the mucus on their surfaces stays
thick and sticky. The cilia, or even coughing, can’t remove it
all
 Symptoms Of Cystic Fibrosis

▪ Thick dehydrated sticky mucus which builds up in

lungs / gut / airways
▪ Infections in lungs causing scars or damage in the
lungs
▪ The mucus prevents secretion (of digestive
enzymes) from pancreas or blocks pancreatic
duct
▪ Malnutrition due to inadequate digestion,
inadequate absorption causing reduced growth
and development
A person with cystic fibrosis is often treated
▪ The mucus blocks sperm duct making males with ‘percussion therapy’ – pummelling
sterile against the back to loosen the thick mucus
so that it can be coughed up
Basic Principles Of Gene Therapy For The
Treatment Of CF
▪ CF is caused by a mutation of the CFTR gene
leading to the formation of a defective CFTR
protein
▪ Treatment involves the insertion of
normal/dominant allele into the DNA of the
affected cells of the respiratory system
▪ Vectors are used to deliver the alleles as an
aerosol spray or inhaler. Viruses and liposomes are
normally used to deliver the alleles
▪ Not all cells take up the virus and the treatment
may have side effects
▪ The effects are short lived, thus treatment needs
repeating
Gene therapy for Leber congenital
amaurosis

▪ A rare form of inherited blindness called Leber

congenital amaurosis (LCA) causes cells in the retina
to die off from an early age
▪ This condition has been reversed using gene therapy
Gene therapy for Leber congenital
amaurosis

▪ The recessive allele causes impaired vision from birth

leading to complete blindness in early adulthood
▪ The dominant allele of the gene, RPE65, codes for a
protein that regenerates visual pigment in rod and
cone cells in the retina after they have been exposed
to light
▪ This allele has been inserted into the cells of people
who are homozygous recessive for this gene
Gene therapy for Leber congenital
amaurosis

▪ Genes can be delivered precisely to the retina, the

layer of light-sensitive cells at the back of the eye
▪ The progress of the treatment can be monitored
easily
▪ Also, there is little activity of immune cells inside the
eye, so there is a low risk of harmful immune
responses to the vector
Gene therapy for SCID
▪ Children with ADA-SCID were often isolated inside
plastic ‘bubbles’ to protect them from infections
▪ Some of the child’s T-lymphocytes are removed and
normal alleles of the ADA gene are introduced into
them, using a virus as a vector
▪ The cells are then replaced
▪ This is not a permanent cure. Regular transfusions
(every three to five months) are necessary to keep the
immune system functioning properly
Gene therapy for SCID
▪ The adeno-associated virus (AAV) is now used as a
vector for inserting the gene into stem cells in bone
marrow
▪ This virus does not insert its genes into the host
genome and so they are not passed on to daughter
cells when a cell divides
▪ Gene therapy of stem cells is more successful than it
was in the 1990s since there is no need for regular
transfusion of T-lymphocytes
 The procedure
for carrying out
gene therapy
for SCID
 Social and ethical ▪ Who decides who should be screened or tested?
considerations of ▪ Which specific disorders should be screened?
using genetic ▪ Who should be providing the screening?
screening and gene ▪ Should we screen or test for disorders for which
therapy in medicine there is no known treatment or cure?
▪ What psychological impact might the results have
on the individuals involved?
▪ Should the results be confidential?
▪ If not, who should be able to have access to the
information?
▪ Should the results be made available to potential
employers, insurers etc?
 Social and ethical
considerations of ▪ DNA analysis is not available for everyone
using genetic
▪ The results may lead to a lifestyle change e.g., Lose
screening and gene weight, stop smoking, take exercise
therapy in medicine
▪ Genetic screening may be helpful in early treatment
▪ It allows people to plan e.g., organise care, appoint
power of attorney, take out medical insurance, make will
or retire early
▪ It may help couples decide whether to have children
▪ The results if positive, may cause anxiety, stress or
depression. If negative, they reduce worry
▪ The results may affect one ability to get insurance, credit
or jobs
Genetically Modified
Organisms In
Agriculture
Insect-resistant crops
 Bt MAIZE

▪ A gene for a toxin, Bt toxin, which is lethal to insects that eat

it but harmless to other animals, has been taken from a
bacterium, Bacillus thuringiensis
▪ Crop plants that contain the Bt toxin gene from B.
thuringiensis produce their own insecticides
 ▪ the evolution of resistance by the insect pests
The most likely
detrimental effects ▪ a damaging effect on other species of insects
on the environment ▪ the transfer of the added gene to other species of
plant
of growing an insect-
resistant crop are:
❖Other disadvantages:
▪ Genetically modified crop seed is expensive and that
its cost may remove any advantage of growing
resistant crops
▪ Growers need to buy seed each season, which again
keeps costs high when compared with those of
traditional varieties.
▪ In parts of the world where a great deal of a
genetically modified crop is grown, there is the
danger of losing biodiversity
The advantages are:

❖Less pesticide is used, reducing the risk of

spray carrying to and affecting non-target
species of insects in other areas
❖Higher yield
Insect-resistance in
cotton plants
▪ Another important agricultural development is that
of GM plants that are protected against attack by
insect pests
▪ Cotton, Gossypium hirsutum, is protected against
pests such as theboll worm
▪ Just like in Bt Maize, yield is improved by genetic
modification
Herbicide-resistant crops
Herbicide-resistant crops

▪ Growing a herbicide-resistant crop allows crop plants to

make full use of the resources available so yields
increase.
▪ The herbicide glyphosate inhibits an enzyme involved in
the synthesis of three amino acids: phenylalanine,
tyrosine and tryptophan
▪ These amino acids are needed to produce proteins
essential for growth. Glyphosate is absorbed by a plant’s
leaves and is then transported to the growing tips
▪ Without the production of proteins in the growth areas,
the plant dies
Herbicide resistance in soybean

▪ Various microorganisms have versions of the

enzyme involved in the synthesis of
phenylalanine, tyrosine and tryptophan that are
not affected by glyphosate
▪ The gene that was transferred into crop plants
came from a strain of the bacterium
Agrobacterium
Herbicide-resistant crops

▪ There are concerns that cultivating crops that are

resistant to herbicides may have detrimental effects
on the environment. These include the following
▪ GM plants are likely to become an agricultural
weed when they grow in fields of other crops
▪ Pollen will transfer the resistance gene to wild
relatives of the crop plant, producing hybrid
offspring that are invasive weeds
▪ Herbicide-resistant weeds will evolve because
so much of the same herbicide is used
 ❑Social implications
▪ GM crops increase food supply
Ethical and social ▪ GM crops decrease food cost
implications of
▪ GM crops increase a country’s wealth
using genetically
modified organisms
(GMOs) in food ❑Ethical implications
production ▪ GM crops relieve hunger or starvation
▪ GM crops reduce land area for crops which enables the
conservation of habitats thus protecting biodiversity