"knockout mouse""knockout mouse"A knockout mouse has had both alleles of a particular gene replaced A knockout mouse has had both alleles of a particular gene replaced

with an inactive allele. This is usually accomplished by using with an inactive allele. This is usually accomplished by using

homologous recombinationhomologous recombination to replace one allele followed by two or more generations ofto replace one allele followed by two or more generations of

selective breeding until breeing pairs are isolated that have both selective breeding until breeing pairs are isolated that have both alleles of the targeted gene inactivated or alleles of the targeted gene inactivated or knocked out.knocked out.

When an investigator wants to replace one allele with an engineeredconstruct but not affect any other locus in the genome, then the method of

choice is homologous recombination. To perform homolgous recombination, youmust 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 ofyour choosing.

The next step is to design and fabricate the DNA construct you want toinsert into the chromosome in place of the wild-type allele. This construct

may contain any DNA sequence of your choosing which means you can insertdifferent alleles (both functional and non-functional ones), different genesor reporter genes (e.g. antibiotic resistance or green fluorescent protein).

Regardless of what you want to insert, you must include some flanking DNAthat is identical in sequence to the targeted locus

In addition to the positive selection marker (e.g. antibiotic resistance) often anegative selection marker (e.g. thymidine kinase, tk) is added to the

replacement vector. The negative marker is outside the region of sequencesimilarity between the vector and the targeted locus.

The engineered construct is added to cells which contain the targeted geneof interest. By mechanisms that are poorly understood but are similar to

what occurrs during meiosis and mitosis when homolgous chromosomes alignalong the metaphase plane, the engineered construct finds the targeted geneand 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 isa new piece of DNA inserted into the chromosome. The rest of the genome is

unaltered but the single targeted locus has been replaced with theengineered 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 surviveThe final product of non-homologous recombination can survivepositive (antibiotic) selection. However, there is a drug called gancyclovirpositive (antibiotic) selection. However, there is a drug called gancyclovir

that will kill any cell that contains the that will kill any cell that contains the tktk gene. So cells undergoing gene. So cells undergoinghomologous recombination are grown in antibiotic to select for recombinationhomologous recombination are grown in antibiotic to select for recombinationand gancyclovir to kill any cells that successfully conducted and gancyclovir to kill any cells that successfully conducted non-homologousnon-homologous

recombination.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 aninactive allele..

Isolate developing embryo at blastocyst stage. This embryo is from a strain of mice with gray fur.

1.

Remove embryonic stem cells from gray-fur blastocyst. Grow stemcells in tissue culture.

2.

Transfect stem cells with homologous recombination construct. Selectfor homologous recombination by growing stem cells in neomycin and gancyclvir.

Implant several chimeric blastocysts into pseudo-pregnant, white furmouse.

Mother will give birth to a range of mice. Some will be normal whitefur 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 bederived 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 gonadsof the chimeric mice were derived from recombinant stem cells, all the

offspring will have gray fur. Every cell in gray mice are heterozygous forthe 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"."knockout mouse".

Knock in módszerEn-1 ko egér: súlyos fejlôdési rendellenességek. En-2 ko egér OK. En-1 kompenzál? És En-2?

Engrailed 1 és 2 egér homeobox gének.

AzEn-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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Granulocita és monocita

B- és T-limfocita

Bone marrow transplantationBone marrow transplantation

+Mx+Mx

+Mx+Mx

-Mx-Mx

-Mx-Mx

irrad. wt rec. ko donor

Az egér füle

+/+ k.o.k.o.k.o. k.o.

Lucerna-tart. táp

Pheophorbide aPheophorbide a is is transported by BCRPtransported by BCRP

Az egér füle

+/+ k.o.k.o.k.o. k.o.

Bone marrow transplantationBone marrow transplantation

Lucerna táp

"knockout mouse""knockout mouse"