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    Types of Cloning Vector

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    Koo Uss Iin

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    © All Rights Reserved
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    Types of Cloning Vector

    Uploaded by

    Koo Uss Iin
    20 views21 pages

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    © © All Rights Reserved

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    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    20 views21 pages

    Types of Cloning Vector

    Uploaded by

    Koo Uss Iin

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 21

    TYPES OF CLONING VECTORS

    CLONING VECTORS
    • Different types of cloning vectors are used for
    different types of cloning experiments.
    • The vector is chosen according to the size and
    type of DNA to be cloned
    PLASMID VECTORS
    Plasmid vectors are used to clone DNA
    ranging in size from several base pairs to
    several thousands of base pairs (100bp -
    10kb).
    CLONING STRATEGY
    Strategy depends on the starting information and
    desired endpoint.
    Starting Information or Resources:
    ▪ Protein sequence
    ▪ Positional cloning information
    ▪ mRNA species / sequence
    ▪ cDNA libraries
    ▪ DNA sequence known or unknown
    ▪ genomic DNA libraries
    ▪ PCR product
    How Are Genes Cloned Using
    Plasmids?
    To understand how genes are cloned, we need
    introduce three terms.
    1. Recombinant DNA- is mixed DNA
    2. Vector -it carries recombinant DNA into
    cells.
    3. Plasmids - are tiny circular pieces of DNA
    that are commonly found in bacteria.
    Why Plasmids are Good Cloning Vectors

    1. small size (easy to manipulate and isolate)


    2. circular (more stable)
    3. replication independent of host cell
    4. several copies may be present (facilitates
    replication)
    5. frequently have antibody resistance (detection
    easy)
    Disadvantages using plasmids
    1. Cannot accept large fragments
    2. Sizes range from 0- 10 kb
    3. Standard methods of transformation
    are inefficient
    BACTERIOPHAGE LAMBDA
    • Phage lambda is a bacteriophage or phage, i.e.
    bacterial virus, that uses E. coli as host.
    • Its structure is that of a typical phage: head, tail, tail
    fibres.
    • Lambda viral genome: 48.5 kb linear DNA with a 12
    base ssDNA "sticky end" at both ends; these ends are
    complementary in sequence and can hybridize to each
    other (this is the cos site: cohesive ends).
    • Infection: lambda tail fibres adsorb to a cell surface
    receptor, the tail contracts, and the DNA is injected.
    • The DNA circularizes at the cos site, and lambda begins
    its life cycle in the E. coli host.
    COSMID VECTOR
    Purpose:
    1. Clone large inserts of
    DNA: size ~ 45 kb
    Features:
    Cosmids are Plasmids
    with one or two Lambda
    Cos sites.
    Presence of the Cos site
    permits in vitro
    packaging of cosmid
    DNA into Lambda
    particles
    COSMID VECTOR
    • Thus, have some advantages of Lambda as
    Cloning Vehicle:
    • Strong selection for cloning of large inserts
    • Infection process rather than transformation for
    entry of chimeric DNA into E. coli host
    • Maintain Cosmids as phage particles in solution
    • But Cosmids are Plasmids:
    Thus do NOT form plaques but rather cloning
    proceeds via E. coli colony formation
    Yeast Artificial Chromosomes
    Yeast Artificial Chromosomes
    Purpose:
    Cloning vehicles that propogate in eukaryotic cell
    hosts as eukaryotic Chromosomes
    Clone very large inserts of DNA: 100 kb - 10 Mb

    Features:
    YAC cloning vehicles are plasmids
    Final chimeric DNA is a linear DNA molecule with
    telomeric ends: Artificial Chromosome
    Additional features:

    1. Often have a selection for an insert


    2. YAC cloning vehicles often have a
    bacterial origin of DNA replication (ori)
    and a selection marker for propogation of
    the YAC through bacteria.
    3. The YAC can use both yeast and bacteria
    as a host
    RETROVIRAL VECTORS
    Retroviral vectors are used to introduce new or altered
    genes into the genomes of human and animal cells.
    Retroviruses are RNA viruses.
    The viral RNA is converted into DNA by the viral reverse
    transcriptase and then is efficiently integrated into the
    host genome
    Any foreign or mutated host gene introduced into the
    retroviral genome will be integrated into the host
    chromosome and can reside there practically indefinitely.
    Retroviral vectors are widely used to study oncogenes
    and other human genes.
    Types of expression systems

    Bacterial: plasmids, phages


    Yeast: expression vectors: plasmids, yeast artifical
    chromosomes (YACs)
    Insect cells: baculovirus, plasmids
    Mammalian:

    viral expression vectors (gene therapy):


    • SV40
    • vaccinia virus
    • adenovirus
    • retrovirus
    Stable cell lines (CHO, HEK293)
    EXPRESSION VECTORS

    Allows a cloned segment of DNA to be


    translated into protein inside a bacterial
    or eukaryotic cell.
    Vectors will contain the ff:
    (a) in vivo promoter
    (b) Ampicillin selection
    (c) Sequencing primers
    EXPRESSION VECTORS
    1. Produces large amounts of a specific
    protein
    2. Permits studies of the structure and
    function of proteins
    3. Can be useful when proteins are rare
    cellular components or difficult to isolate
    To produce the product of a cloned gene for further
    Expression vectors studies
    1) Expression vectors with a strong promoter
    More mRNA More protein

    2) Expression vectors with an inducible promoter


    Foreign proteins when overexpressed could be toxic
    Keep the gene expression off till it is time to turn it on

    a. Drug-inducible (e.g. IPTG or arabinose)


    b. Heat-inducible
    3) Expression vectors with a fusion tag for affinity purification
    Facilitate the purification of the expressed protein
    • 6 Histidine tag
    • Glutathione transferase tag (GST)
    • Maltose-binding protein tag
    Common problems with bacterial
    expression systems
    Low expression levels:
    ▪ change promoter
    ▪ change plasmid
    ▪ change cell type
    ▪ add rare tRNAs for rare codons on second plasmid
    Severe protein degradation:
     use proteasome inhibitors and other protease inhibitors
     try induction at lower temperature
    Missing post-translational modification: co-express with kinases etc.
    Glycosylation will not be carried out:
     use yeast or mammalian expression system
    Misfolded protein (inclusion bodies):
     co-express with GroEL, a chaperone
     try refolding buffers
    REPORTER GENE VECTORS

    A gene that encodes a protein whose activity


    can be easily assayed in a cell in which it is
    not normally expressed
    These genes are linked to regulatory
    sequences whose function is being tested
    Changes in transcriptional activity from the
    regulatory sequences are detected by
    changes in the level of reporter gene
    expression
    SHUTTLE VECTORS
    Shuttle vectors can replicate in two
    different organisms, e.g. bacteria and
    yeast, or mammalian cells and bacteria.
    They have the appropriate origins of
    replication.
    Hence one can clone a gene in bacteria,
    maybe modify it or mutate it in bacteria,
    and test its function by introducing it into
    yeast or animal cells.

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