This procedure is required for mouse ES cells imported from other institutions as well as for mouse ES cells targeted by the Core prior to transfer into blastocysts to generate chimeric mice. At least twenty chromosome spreads will be prepared and chromosomes will be counted to determine the percentage of euploid cells.
When generating chimeric mice using targeted mouse ES cells, the goal is to make chimeric mice that can transmit the desired genetic modification to their offspring and create a line. At this point in time, there are only a few indicators (not guarantees) of the likelihood that a chimera may or may not transmit.
The primary pre-injection predictors of germline potential are all related to the maintenance of the mouse ES cells in a pluripotent state. Mouse ES cells need to be of a low passage number, should be mycoplasma-free, and a large percentage of them should be euploid, as determined by chromosome counting. We ensure that all parental mouse ES cells used in the Core are of a low passage number, have a high percentage of euploidy, and are mycoplasma-free. Following gene targeting, it is critical to reassess the euploidy of your recombinant clones. We will inject ES clones with greater than 60% euploidy, although we strongly encourage investigators, when they have the option, to inject those clones with more than 70% euploidy. Clones with less than 60% euploidy can be subcloned to try to isolate clones with improved euploidy.