Knockout Mouse Creation

What happens when we create deliberate mutations in a particular gene in a living organism?

We now have techniques for targeted disruption of genes in several organisms. Most popular organism used for these targeted gene disruptions are mice.

A knockout mouse or transgenic animal is generated by replacing both alleles of a particular gene with an inactive allele. This is done by using homologous recombination to replace one allele which is then followed by two or more generations of selective breeding until a breeding pair is isolated that has both alleles of the targeted gene knocked out. These knockout mice allow researchers to determine the role of a particular gene by observing the phenotype of individuals that lack the gene completely.

Steps involved in making a Knockout Mouse:

1) Developing embryo at blastocyst stage is first isolated. This embryo is from a strain of mice with gray fur.

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

3) Transfect stem cells with homologous recombination construct. Select for homologous recombination by growing stem cells in neomycin and gancyclvir.

4) Remove homologously recombined stem cells from petri dish and inject into a new blastocyst that would have only white fur.

5) Implant several chimeric blastocysts into pseudo-pregnant, white fur mouse.

6) 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.

7) 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.

8) 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".