Download BIL 250 - Knockout Mouse

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BIL 250 -‐‑ Knockout Mouse
Knocking out a Gene
Step One: isolating a "knocked out" gene in
cell culture
A cloned gene of interest is taken from a library
It is inactivated via insertion of a known gene (the one used in the
example here confer resistance to neomycin, which is lethal to cells
not carrying the neor gene)
The vector also is given another marker, this one called "tk", which
makes any cell carrying it lethally sensitive to the drug ganciclovir.
(The "tk" gene has a restriction site that excludes it if the neomycin
resistance-inactivated vector is inserted into the desired location,
replacing the wild type gene of interest)
Embryonic Stem Cells (ES cells) from a wild type agouti mouse are
cultured in vitro
Vectors are added to the mouse cell culture and the scientist
crosses his/her fingers
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BIL 250 -‐‑ Knockout Mouse
Three things could happen:
Vector will insert into the desired target gene (in this case, it's
the Mystery Gene "M", whose function is unknown), replacing it
with the mutant (inactivated) version (with the neor insertion)
Vector will insert ectopically (in a random location)
Vector won't insert at all
The desired result, of course, is replacement of the wild type
gene (M) with the artificially knocked out version (m). To isolate
cells in which replacement has successfully taken place:
Putatively transfected cells from your suspension are grown in
vitro.
The agar medium contains both neomycin and gangclovir.
Hence,
neomycin will kill the cells that don't have a neor insertion (the
knocked out "m" allele)
gangclovir will kill the cells that have ectopic insertions, as those
will have retained the "tk" gene.
This leaves only the cells that have the insertion of the mutant,
inactivated form replacing the wild type gene of interest.
These successfully transfected cells can be cloned and used for
further study.
Step Two: Creating a Chimera Mouse
(carrying the knocked out gene)
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BIL 250 -‐‑ Knockout Mouse
Your cell culture of diploid cells now contains one normal
chromosome and one with a knocked out version of the wild
type.
These modified Embryonic Stem (ES) cells from the wild type
agouti mouse are injected into a blastocyst from a strain of
black mice (black fur is a recessive version of the A gene,
coding for agouti fur).
The modified embryos are inserted into a surrogate mother
mouse, who gives birth to them.
The baby mice who have patches of both black and agouti fur
are the chimeras who have grown from embryos that
successfully incorporated the modified Embryonic Stem Cells
from the agouti mice.
Choose a male chimera for greatest reproductive output.
Step Three: Breeding Knockout Mice
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BIL 250 -‐‑ Knockout Mouse
If you mate the male chimera to a black female, 50% of the
offspring should carry the knocked out gene from the father
mouse.
By taking tissue samples, isolating DNA and examining it directly
for evidence of the insert, the individuals carrying the knocked
out gene can be identified.
These Mm heterozygotes can then be mated together.
According to Mendel's Law, 25% of the resulting offspring will be
mm.
The knocked out gene may not have an obvious phenotypic
expression, in which case the knockout mouse DNA can be
examined directly for evidence of homozygous recessive
condition.
In the example above, however, the knocked out gene
resulted in a curly tail in the homozygous knockout mice: The M
gene must have something to do with normal development of
the caudal vertebrae in wild type mice.
And so it goes.
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