Gene Targeting and Knockout Mouse Production

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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.