Download homologous recombination

"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.
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
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 .
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
recombination.
The positive and negative selection markers are incorporated into chromosome
so gancyclovir will kill cells with modified chromosomes.
A knockout mouse has had both alleles of a particular gene replaced with an
inactive allele..
1.
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.
2.
Transfect stem cells with homologous
recombination construct. Select
for homologous recombination by
growing stem cells in neomycin and
gancyclvir.
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.
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".
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?
Az
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.
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Bone marrow transplantation
-Mx
Granulocita
és monocita
+Mx
irrad. wt rec.
ko donor
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B- és Tlimfocita
-Mx
+Mx
Az egér füle
+/+
k.o.
Pheophorbide a is
transported by BCRP
Lucerna-tart. táp
k.o.
k.o.
k.o.
Az egér füle
+/+
k.o.
k.o.
k.o.
Bone marrow transplantation
Lucerna táp
k.o.
"knockout mouse"