knockout mouse

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"knockout mouse"
A knockout mouse has had both alleles of a
particular gene replaced with an inactive
allele. This is usually accomplished by using
homologous recombination to replace one allele
followed by two or more generations of selective
breeding until breeing pairs are isolated that
have both alleles of the targeted gene
inactivated or knocked out.
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When an investigator wants to replace one allele
with an engineered construct but not affect any
other locus in the genome, then the method
of choice is homologous recombination. To perform
homolgous recombination, you must know the DNA
sequence of the gene you want to replace.
With this information, it is possible to replace
any gene with a DNA construct of your choosing.
The next step is to design and fabricate the DNA
construct you want to insert into the chromosome
in place of the wild-type allele. This
construct may contain any DNA sequence of your
choosing which means you can insert different
alleles (both functional and non-functional
ones), different genes or reporter genes (e.g.
antibiotic resistance or green fluorescent
protein). Regardless of what you want to insert,
you must include some flanking DNA that is
identical in sequence to the targeted locus
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In addition to the positive selection marker
(e.g. antibiotic resistance) often a negative
selection marker (e.g. thymidine kinase, tk) is
added to the replacement vector. The negative
marker is outside the region of
sequence similarity between the vector and the
targeted locus. The engineered construct is added
to cells which contain the targeted gene of
interest. By mechanisms that are poorly
understood but are similar to what occurrs during
meiosis and mitosis when homolgous chromosomes
align along the metaphase plane, the engineered
construct finds the targeted gene and
recombination takes place within the homolgous
(meaning identical in this case) sequences.
Once the cells have performed their part of the
procedure, the end result is a new piece of DNA
inserted into the chromosome. The rest of the
genome is unaltered but the single targeted locus
has been replaced with the engineered construct
and some of its flanking DNA .
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If the targeting vector aligns in a
non-homologous region of the genome, then
recombination is random and the negative
selection marker may become incorporated into the
genome.
The final product of non-homologous recombination
can survive positive (antibiotic) selection.
However, there is a drug called gancyclovir that
will kill any cell that contains the tk gene. So
cells undergoing homologous recombination are
grown in antibiotic to select for
recombination and gancyclovir to kill any cells
that successfully conducted non-homologous recombi
nation.
The positive and negative selection markers are
incorporated into chromosome so gancyclovir will
kill cells with modified chromosomes.
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A knockout mouse has had both alleles of a
particular gene replaced with an inactive allele..
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2.
Isolate developing embryo at blastocyst stage.
This embryo is from a strain of mice with gray
fur.
Transfect stem cells with homologous
recombination construct. Select for homologous
recombination by growing stem cells in neomycin
and gancyclvir.
Remove embryonic stem cells from gray-fur
blastocyst. Grow stem cells in tissue culture.
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Implant several chimeric blastocysts into
pseudo-pregnant, white fur mouse.
Mother will give birth to a range of mice. Some
will be normal white fur mice but others will be
chimeric mice. Chimeric mice have many of
their cells from the original white fur
blastocyst but some of their cells will
be derived from recombinant stem cells. Fur cells
from recombinant stem cells produce gray patches
which are easily detected.
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Mate the chimeric mice with wild-type white fur
mice. If the gonads of the chimeric mice were
derived from recombinant stem cells, all
the offspring will have gray fur. Every cell in
gray mice are heterozygous for the homologous
recombination.
Mate heterozygous gray mice (/ H) and genotpye
the gray offspring. Identify homozygous
recombinants (H / H) and breed them to produce a
strain of mice with both alleles knocked out. The
pure breeding mouse strain is a "knockout mouse".
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Knock in módszer
Engrailed 1 és 2 egér homeobox gének.
En-1 ko egér súlyos fejlôdési rendellenességek.
En-2 ko egér OK. En-1 kompenzál? És En-2?
En-2 az En-1 promoter kontrollja alá került. En-1
hamarabb expresszálódik, mint En-2. Az En-1/En2
knock in egér OK, tehát az En-2 képes
helyettesíteni az En-t.
Az
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Bone marrow transplantation
Granulocita és monocita
irrad. wt rec. ko donor
B- és T- limfocita
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Az egér füle
k.o.
k.o.
k.o.
k.o.
/
Pheophorbide a is transported by BCRP
Lucerna-tart. táp
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Bone marrow transplantation
Lucerna táp
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"knockout mouse"