Due to the increased production of C57BL/6 derived ES cells for the generation of
knockout mice, selecting germline competent chimaeras by coat colour chimaerism is
no longer a reliable method, since even highly agouti C57BL/6 chimaeras can fail to
transmit the manipulated allele. In genotyping sperm cell DNA, chimaeras with ES cell
contribution to their germline, can be identified and subsequently used for matings.
Chimaeras used in this study were mated with C57BL/6Tac mice. Mating results and
the estimated degree of coat colour chimaerism was compared to the results obtained
in the neomycin genotyping PCR.
Sperm cell samples were collected either by separating mice during ejaculation and/or
by transepidermal sperm cell aspiration. For sperm cell DNA extraction a precipitation
method was used, as well as a binding column protocol. The extracted samples were
used as a template for the genotyping PCR.
All chimaeras, which had transmitted the gene of interest in matings showed a neo
signal in PCR analysis. Chimaeras, which did not show a neo signal, also had not
transmitted in matings.
Consequently, chimaeras, not showing a signal for the induced mutation in their sperm
cell samples, can be excluded from breeding.
Identification of germline competent chimaeras by spermatozoa genotyping prior to
breeding will lead to considerable savings in mouse
numbers and resources.