Knockout mouse production is a combined effort with the principal investigator and the Knockout Mice Core. The principal investigator’s responsibilities include designing and building the targeting construct, as well as the strategy for the Southern blot genotyping. Consulting the Knockout Mice Core on these steps may help avoid common problems and speed up the process. The facility will then electroporate the embryonic stem (ES) cells (129SvEv or C57-BL6), select cell lines for genotyping and extract DNA for testing. Positive clones are then prepared for use and frozen for storage. Targeted ES cells are then injected into C57-BL6 albino blastocysts and transferred to recipient foster CD1 female mice.
The result of this will be pups containing the genetic material from both the C57-BL6 albino and the targeted ES cells in a 129 or B6 background. These chimeric male mice will be grown to breeding age and mated with C57-BL6 albino female mice. The pups produced from this breeding come from gametes that originated from the ES cells. Their coat color will be either agouti or black, depending on the ES cell used, while germ cells from the blastocyst portion will produce white pups. This coat color marker system reduces the number of pups to be genotyped considerably.
When a chimera produces a litter containing agouti or black pups, the pups are tested by Southern analysis to determine the presence of the targeted gene. If the Southern strategy is done correctly, and is repeatable, 50% of these agouti pups should carry the modified gene in question. If however, the Southern strategy is poor, one may never find the knockout mouse they are looking for.