Gene Targeting

Gene-targeted mice, called “knock-out” or “knock-in” mice, are produced by injecting blastocysts with embryonic stem (ES) cells that have been genetically modified. ES cells are pluripotent stem cells that are derived from the inner cell mass of 3.5-dpc embryos called blastocysts. These undifferentiated cells have the capacity to differentiate into most tissues of the mouse.

Typically, 129/SvJ strain ES cells are transfected by electroporation with an isogenic DNA construct containing homology to a region of the genome. By a process called homologous recombination, this DNA fragment will insert into the homologous gene either “knocking out” the function of the gene or “knocking in” a functional gene into the same locus.

The transfected cells are placed in a selective media and the surviving clones are isolated, grown in culture, replicated, and placed in frozen storage. The cells of one replicate are processed for DNA isolation and the clones testing positive for homologous recombination are thawed and expanded. These positive clones are injected into blastocysts from a donor mouse and implanted into pseudopregnant foster mothers. Chimeric offspring are backcrossed to test for germline transmission of the targeted gene. Heterozygous mice are then intercrossed to derive homozygous gene-targeted mice.