In-vitro Mutagenesis (Gene Knockouts)

Scientists can inactivate, or “knock out,” a gene of interest in mice to study its function. This is usually done by replacing the gene with a targeting vector, which is an engineered DNA sequence.

The targeting vector contains sequences homologous to the regions before and after the gene. These matching regions are called homology arms.

The targeting vector also contains marker genes that help identify cells in which correct gene replacement has happened.

These include a positive selection marker, such as the neomycin resistance gene, NeoR, located in the middle of the targeting vector. This marker provides resistance to the antibiotic neomycin.

A negative selection marker, such as the thymidine kinase gene, or TK, is located outside the homologous region. This marker makes cells sensitive to the drug ganciclovir.

When introduced into mouse embryonic stem cells, the vector replaces the target gene through homologous recombination between its homology arms and the matching DNA sequences in the genome.

After homologous recombination, the target gene is replaced by the NeoR gene, while the TK gene is not incorporated because it lies outside the homologous region.

As a result, cells that undergo correct homologous recombination contain NeoR but not TK. These cells survive treatment with both neomycin and ganciclovir, allowing researchers to select them in culture.

These cells are then injected into early-stage mouse embryos, which are implanted into the uterus of a female mouse. The resulting mice are chimeric because their tissues contain cells derived from both the original embryo and the genetically modified embryonic stem cells.

When the chimeric mouse is bred, some offspring inherit one copy of the knockout allele and are called heterozygous knockout mice.

Breeding these mice can produce homozygous knockout mice, in which both copies of the target gene are inactivated.