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Gene Targeting and Knockout Mouse Production

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Gain of function: transgenics, gene repair. Loss of function: (knockout, knockdown, ... (agouti) agouti black. Is this a useful chimera? YES!. Acknowledgements ... – PowerPoint PPT presentation

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Title: Gene Targeting and Knockout Mouse Production


1
Gene Targeting and Knockout Mouse Production
  • MCD Biology Presentation
  • 10 Feb, 2009

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Gene Targeting and Transgenic Technologies
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Find the right poison, make the right mutant
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Three flavors
  • Gain of function transgenics, gene repair.
  • Loss of function (knockout, knockdown, and
    conditional.)
  • Neutral i.e. knockin (reporter assay), tissue
    specific Cre recombinase expression.
  • Expression of a reporter linked to endogenous
    gene function.

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Two strategies
  • Manipulate cells in vitro and transfer them into
    an early embryo.
  • Produces a chimera (part to whole)
  • Introduce DNA directly into a fertilized oocyte
    (in vivo.)
  • Theoretically produces a hemizygous mouse with a
    copy of the gene in EVERY cell.
  • Usually over-expressed, can be tissue specific.

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Vectors, Stem Cells and Gene Targeting
  • Gene targeting vectors
  • Vectors are a mechanism for inserting DNA into
    chromatin.
  • Two basic designs
  • Replacement vectors interrupts or deletes and
    replaces coding region of a gene (most common.)
  • Insertion vectors

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Replacement Vectors
  • Contains positive selection marker, homologous
    sequence (targeting portion), linearization site
    and plasmid backbone. May also contain negative
    selection marker.
  • Positive selection marker is used to identify
    cells that have undergone a successful
    transfection.
  • Homologous sequence is isogenic to wild type gene
    locus.
  • Linearization site is necessary for
    recombination. Can be manipulated to reduce
    reversible integration.
  • Negative selection marker is used to select for
    clones that only have targeted insertions.

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Replacement Vectors or Generation of Knockouts
(null allele.)
  • Oversized insertion at 5 locus disrupts gene.
  • Must be familiar with RNA splicing, insertion may
    be spliced out of final transcript.
  • Introduction of a stop codon early in coding
    sequence.
  • May produce a truncated transcript
  • Deletion or insertion of base pairs to create
    frameshift mutation.
  • All methods may create secondary phenotypes by
    producing truncated proteins or perturbing
    activation of downstream alleles.
  • WT compensation for gene ablation.

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Replacement Vector Interruption of targeted
allele (knockout.)
Plasmid Backbone
Homologous Sequence
Positive Selection Marker
Targeting Vector
Exon
Negative Selection Marker
Wild type allele
Result Integration
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Positive Selection Elements
  • Positive integration confers resistance to drug
    treatment. Kills or delays proliferation of
    cells that do not contain transcript.
  • Typically dominant selectable markers are used
    because they are rare in ES cells.
  • G418 (neo), Puromycin, Hygromycin B and
    Mycophenolic acid.

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Negative Selection Markers
  • Placed outside sequence of homology to detect
    integration of plasmid backbone.
  • Kills cells that have random integrations of
    entire vector sequence.
  • 6TG (Glancovir), FIAU
  • After positively selected cells are replated and
    amplified, TX with negative selection drug kills
    cells with random integrations.

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Site Specific Recombination
  • Creates specific recombination sites that are
    activated by a recombinase. Faithful excision
    without gain or loss of single base pair.
  • 34 bp. recognition site, orientation specific.
  • Two most common types are Cre/loxP (prokaryotic
    origin) and Flp/FRT (eukaryotic origin.)
  • Two step process
  • Standard replacement vector with addition of loxP
    or FRT sites surrounding sequence that you wish
    to remove/ replace.
  • Transfection with plasmid containing
    recombination enzyme (and conditional promoter/
    enhancer sequence if desired) or breeding against
    tissue specific Cre/Flp mouse.
  • Composed of two elements recombinase enzyme and
    DNA sequence recognized by the recombinase enzyme.

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Some Applications for recombinase mediated
mutations
  • Deletions
  • Removal of selection marker genes
  • Swapping sequences
  • Conditional gene (in)activation
  • Inversions
  • Conditional gene expression
  • Human (inversion) disease models
  • Translocations
  • Human (translocation) disease models

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Plasmid Backbone
Homologous Sequence
Positive Selection Marker
Targeting Vector
Negative Selection Marker
Wild type allele
Result Integration
Positive Selection Marker
Cre/Flp
Positive Selection Marker

Result Deletion
loxP or FRT site
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Part II. Embryo Manipulation
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Now what?
  • ES cells with desired mutation must be introduced
    into the germline in vivo.
  • Two basic methods shake and bake (aggregation)
    and microinjection (MI).
  • Aggregation inexpensive, excellent for embryonic
    lethal mutations.
  • Microinjection expensive, requires microsurgical
    skills.
  • At UCSC we utilize both methods for different
    reasons.

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Aggregation vs. Microinjection
  • Aggregation combines a compact morula with ES
    cells in an enclosed environment.
  • ES cells fuse with host embryo and migrate to
    various tissues.
  • Microinjection involves direct, surgical transfer
    of ES cells into the interior of a blastocyst.
  • ES cells fuse directly with cells from the inner
    cell mass of the embryo with very high
    efficiency.
  • ES cells migrate to gonads, offspring are 100
    mutant.

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Basic Steps
  • Create ES line and select for targeted clones.
    Amplify. (Start months in advance.)
  • Induce ovulation in mice, harvest embryos at E3.5
    (blastocysts.)
  • Generate surrogate mothers (pseudo pregnant)
    mate females against vasectomized males.
  • Delay by one day to compensate for culture
    conditions and damage to embryos during
    microinjection.

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Gene Targeting, /- selection and amplification
of targeted clones.
In Vitro Studies
In Vivo (phenotypic Studies
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Basic Steps
  • Prepare ES cells by treating with trypsin to
    create single cell suspension and inject cells
    into host embryo.
  • Transfer into pseudo pregnant mice.
  • Score and mate high percentage chimeras to
    generate F1 (heterozygous) progeny.
  • Intercross F1s to produce homozygous mutants.
  • Study phenotype.

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What this really looks like
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ES Cell Transfer by Microinjection
(Hogan, et. Al.)
Inner Cell Mass
Trophectoderm
ES Cells
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Normal C57BL/6 Blastocyst (black)
ICM
agouti black
ES cells 129/SvJ (agouti)
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Is this a useful chimera?
YES!
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Acknowledgements
  • A. L. Joyner (ed.) (2000) Gene Targeting, A
    Practical Approach 2nd ed., Oxford University
    Press.
  • B. Hogan, et. Al. (1994) Manipulating the Mouse
    Embryo 2nd ed., Cold Spring Harbor Laboratory
    Press.
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