Generation of transgenic rats by ooplasmic injection of sperm cells exposed to exogenous DNA

Intracytoplasmic sperm injection (ICSI) has been successfully achieved in mice and rats using a piezo-driven injection pipette, with the offspring rate of >30%. The ICSI technique was applied not only to rescue infertile male strains but also to produce transgenic rodents. The ICSI-mediated DNA transfer, that the sperm heads and exogenous DNA solution are mixed and co-injected into ooplasm, has been equally effective to the conventional pronuclear DNA microinjection. Production efficiency of transgenic founders by the ICSI-mediated DNA transfer was comparable between mice and rats, while the optimal DNA concentration was lower in rats than mice. The production efficiency was improved when membrane structure of sperm heads was partially disrupted by detergent or ultrasonic treatment before exposure to the exogenous DNA solution. Exogenous DNAs with various chain lengths were stably integrated into the rodent genomes of various genetic backgrounds by this method. The ICSI-mediated DNA transfer in which the preparation of pronuclear-stage fertilized zygotes is not required would be alternative to conventional pronuclear DNA microinjection.