Genetic Manipulation in the Mouse

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Genetic Manipulation in the Mouse

• Laura Wilding
• laura.wilding_at_vanderbilt.edu
• Gannon Lab

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Helpful Websites

• http//www.bio.davidson.edu/courses/genomics/met
  hod/homolrecomb.htmlhc
• http//users.rcn.com/jkimball.ma.ultranet/Biolog
  yPages/T/TransgenicAnimals.html
• http//www.biochem.arizona.edu/classes/bioc471/pa
  ges/Lecture19/Lecture19.html

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Problem Your PI cloned a gene (YFG) and you can
only graduate when you find out

• How is YFG regulated in vivo?
• What is the result of inactivating YFG?
• What is the role of YFG in the liver?

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How do you determine how YFG is regulated?
Another lab that studies YFG showed that a
promoter fragment drives expression of a
reporter gene in liver cell culture
regulatory elements
txn. start
4.5 kb
How do we determine if the same is true in vivo?
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Two different approaches have been developed to
study gene function in vivo

• DNA microinjection into single cell embryos to
  produce
• transgenic mice containing one or more copies of
  randomly
• integrated expression vector DNA. (Transgenic)
• 2) Use of homologous recombination at defined
  genomic
• loci to create gene knockouts (KOs) or
  replacements.

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How do you determine how YFG is regulated?
Create a transgenic mouse
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Building a transgene

• Eukaryotic genes need
• 1) A promoter/enhancer that will drive
  expression in the tissue of interest
• 2) An open reading frame to encode the protein
  you want to express
• 3) Sequences that ensure correct mRNA processing
  
• a. polyadenylation signal AAUAAA
• b. intron

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Building a transgene
Isolate cDNA coding for the protein you want to
express
Restriction enzymes
plasmid
most genes are now available in plasmids
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Building a transgene
Isolate cDNA coding for the protein you want to
express
Incorporate the promoter/enhancer elements you
want to drive expression
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Building a transgene
Isolate cDNA coding for the protein you want to
express
Incorporate the promoter elements you want to
drive expression
Ensure correct RNA processing
constructed in plasmid
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Building a transgene
Isolate cDNA coding for the protein you want to
express
Incorporate the promoter elements you want to
drive expression
Ensure correct RNA processing
Remove from plasmid
constructed in plasmid
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Once your construct is made, it is sent to the
Transgenic Core here at Vanderbilt
Vanderbilt Transgenic Core
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DNA microinjection into single cell embryos
Fertilized one-cell eggs for DNA microinjection
are obtained from superovulating female mice
that have been mated to a male
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DNA microinjection into single cell embryos
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DNA microinjection into single cell embryos
Using a micromanipulator, a very fine tipped
pipette is inserted into the cytoplasm or
nucleus. The transgene construct is injected
directly into the single cell embryo.
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Implanting microinjected eggs
19 days
Following microinjection, 20-30 eggs are
implanted into the oviduct of a pseudopregnant
surrogate female leading to the production of
5-8 live pups 19 days later.
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Identifying Transgenic Mice
3 weeks
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Establishing founder lines

• Mice that are positive by PCR analysis are
  backcrossed
• to non-transgenic mates

Transgene was silenced and M4 is not a founder
Option 1 pups from the backcross have the
transgene--gt the transgene was integrated into
the germline and mouse is a founder Option 2
no pups from the backcross have the transgene--gt
the transgene was not passed on
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Determining where your transgene is expressed
(i.e- where can your promoter/enhancer drive
expression of the reporter)
brain
X-gal stained
e9.0
liver
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Concerns when making transgenic mice

• Choice of promoter
• How well is your promoter of choice
  characterized?
• Random Insertion
• DNA inserts randomly into the genome
• Consequences
• Subject to effects of surrounding DNA elements
• Multiple copies could insert in tandem
• Results could be due to transgene copy number
• Must screen several lines
• Ectopic expression
• Expression of your transgene where your gene is
  not normally expressed

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Problem Your PI cloned a gene (YFG) and you can
only graduate when you find out

• How is YFG regulated?
• What is the result of inactivating YFG?
• What is the role of YFG in the liver?

22
Two different approaches have been developed to
study gene function in vivo

• DNA microinjection into single cell embryos to
  produce
• transgenic mice containing one or more copies of
  randomly
• integrated expression vector DNA.
• 2) Use of homologous recombination at defined
  genomic
• loci to create gene knockouts (KOs) or
  replacements.

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What is the effect of inactivating YFG?
Use of homologous recombination at defined
genomic loci to create gene knockout (KOs).
Creating the targeting construct Deriving
ES cells Creating the KO mouse using ES cells
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Creating the targeting construct
YFG
What components do we need to knockout YFG?
- a mutation in YFG to render it inactive
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Creating the targeting constructWhat we WANT to
happen
HR
Endogenous locus with mutation in YFG and a
neomycin resistance cassette
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Creating the targeting construct What can (and
will) happen
The cells (the majority) that failed to take up
the vector
In a few cells the vector is inserted randomly
in the genome.
In still fewer cells homologous recombination
occurs.
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Deriving ES cells
Isolate developing embryo at blastocyst stage.
This embryo is from a strain of mice with gray
fur.
Remove embryonic stem cells from gray-fur
blastocyst. Grow stem cells in tissue culture.
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Creating the KO mouse using ES cells
ES cells
Outcomes-
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Culture the mixture of cells in medium containing
both G418 and gancyclovir.
Creating the KO mouse using ES cells
no G418 resistance DIE
Contain tk gene- DIE from gancyclovir
LIVE
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Creating the KO mouse using ES cells
Remove homologously recombined stem cells from
petri dish and inject into a new blastocyst from
an animal that would have only white fur.
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Creating the KO mouse using ES cells
Implant several chimeric blastocysts into
pseudo-pregnant, white fur mouse.
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• Mother will give birth to a range of mice.

Option 1 pup is white --gt all cells came from
white furred mother Option 2 pup is chimeric
(gray and white) --gt some cells are derived from
white furred mother, some from gray furred
recombinant stem cells
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• Mate the chimeric mice with wild-type white fur
  mice.

Option 1 all pups are gray --gt therefore,
gonads were derived from recombinant stem cells
(germline transmission) Option 2 none of pups
are gray --gt no germline transmission of
recombined allele
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• Mate heterozygous gray mice (/ H) and genotype

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Detection of Homologous Recombination (Example)
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Problem you want to know how YFG functions in
the liver, but since your gene is expressed in
the brain as well, a KO of YFG is embryonic
lethal
What can you do??
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What if your KO is embryonic lethal??
brain
X-gal stained
e9.0
liver
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What if your KO is embryonic lethal??
never born
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Conditional InactivationCre-Lox Technology
Cre is a 38 kDa recombinase protein from
bacteriophage P1 which mediates recombination
between loxP sites loxP sites A loxP site
consists of two 13 bp inverted repeats separated
by an 8 bp asymmetric spacer region
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Cre-Lox Technology- how does it work?
Two loxP sites are inserted on each side of an
essential exon of the gene of interest by
homologous recombination. These sites must not
disrupt gene function.
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Cre-Lox Technology- Application in the mouse

• Need 2 different mice

For our problem, we can use the albumin promoter
to drive the expression of Cre because that is
expressed in the liver but not in the brain
THEREFORE
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Liver Specific Gene Inactivation
In the liver we have Cre expression and this
allows for the excision of our gene
In all other cell types there is no Cre
expression thus our gene function remains normal
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Concerns for making a KO mouse

• No phenotype-
• Gene compensation
• Time and money (not always unlimited)
• At least 18 months to make a KO mouse
• 3,000-5,000 dollars to make a KO