Cryopreservation of Gene Disrupted Mouse Spermatozoa

Abstract

Cryopreservation of mouse spermatozoa has recently become available for use. In this study, we attempted to apply this technique to the maintenance of gene disrupted mouse lines. Cauda epididymides that were taken from Et1^tm1Csk and Gk^tm1Csk mice were minced in 100 μl of a cryopreservation solution which consisted of 18% raffinose and 3% skim milk. Sperm suspensions in plastic straws were cooled rather rapidly by being placed in the gas phase above liquid nitrogen, and then were stored at -196°C. The frozen straws were thawed rapidly being by immersing in a water bath at 30°C. For in vitro fertilization, 1 μl of thawed sperm suspensions from each mouse strain was added directly to oocytes contained in 200 μl of medium. Following pre-incubation of the frozen-thawed spermatozoa, ICR oocytes were introduced into the medium containing the frozen-thawed spermatozoa. The fertilization rates of the oocytes inseminated with Et1^tm1Csk and Gk^tm1Csk frozen-thawed spermatozoa were 71% and 77%, respectively. The development rates into young after embryo transfer were in a range of 32-65%. Progeny tests revealed that the mutated locus/loci were transmitted to the next generation according to the Mendelian law of the inheritance of characteristics. These results indicate that cryopreservation of spermatozoa provides an effective alternative to embryo freezing for maintenance of gene disrupted mouse strains.