CONICET | Buscador de Institutos y Recursos Humanos

The transfer of genetically modified mice from one Animal Facility to another is not allowed if the animals to be transferred have pathogens different from those present in the recipient facility. Recently, a research group requested our service to introduce a BALB/C mouse line carrying the desired gene modification in homozygosis into a new facility. For this purpose, the method chosen was to first perform in vitro fertilization using spermatozoa from the genetically modified males followed by uterine transfer of the in vitro generated blastocysts into pseudopregnant wild type recipients. For this purpose, epididymal sperm were recovered from a donor male of proven fertility and subjected to in vitro capacitation in fertilization medium (HTF). On the other hand, 5 prepuberal wild type BALB/C females were subjected to ovarian superstimulation with injections of eCG and hCG separated by 48 hours. Twelve hours after hCG administration, the cumulus-oocytes complexes were collected from the females and inseminated. Gametes were co-incubated in HTF until eggs reached the 2-cell stage. The resulting 71 2-cell embryos (64% fertilization rate) were incubated in an appropriate medium (KSOM) supplemented with amino acids for embryonic development. The 14 blastocysts (20% embryo development rate) recovered after 4 days of culture were transferred into a pseudopregnant female (previously mated with a vasectomized male) using a commercial device (NSETTM) without the need of surgery or anesthesia. Three weeks later, 3 BALB/C pups were born in the new facility (21% live born rate). PCR genotyping showed that all pups carried the desired transgene in heterozygosis. To our knowledge, these results showing the successful recovery of a mouse line from sperm of a male housed in an external Facility represent the first case reported so far in our country.