Chimera rescue by sperm cryopreservation, genotyping and IVF or ICSI:
Production of genetically modified mouse lines is dependent on successful germline transmission (GLT) of the mutant allele from productive chimera. When natural mating fails to achieve heterozygous mice due to infertility of the male chimera, chimera illness, or other obstacles, sperm may be harvested from chimeras, genotyped by Taqman PCR to determine the relative mutant allele contribution, and sperm quality assessed prior to specialized artificial reproductive techniques (in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI)). Using both genotyping and sperm quality assessment, sperm from the best chimera may be selectively utilized for IVF or ICSI to rescue germline transmission. Here at the MBP, we have successfully rescued numerous projects by producing heterozygous mice from sperm collected from non-productive chimeras.
Li MW, Willis BJ, Evans KD, Araiza RS, Lee AY, Lloyd KC. Rescue of germline transmission from chimeras by IVF after sperm analysis. Transgenic Res 24:99-108.