Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live Rhesus monkey births

Exogenous DNA transfer, mediated by intracytoplasmic sperm injection (ICSI) with plasmid-bound spermatozoa, results in the production of transgene expressing embryos in rhesus macaques (Macaca mulatta, mean = 34.6%; n = 81). Rhodamine-tagged DNA encoding the green fluorescent protein (GFP) gene binds avidly to spermatozoa. The rhodamine signal, while lost at the egg surface during in-vitro fertilization (IVF), is traced by dynamic imaging during ICSI and remains as a brilliant marker on the microinjected spermatozoa within the oocyte cytoplasm. The transgene is expressed in preimplantation embryos produced by ICSI, but not IVF, as early as the 4-cell stage with the number of expressing cells and the percentage of expressing embryos increasing during embryogenesis to the blastocyst stage. The three offspring that resulted from seven embryo transfers (a set of anatomically normal twins, one male and one female, stillborn 35 days premature, and a healthy male born at term) demonstrate that primate spermatozoa with exogenously bound DNA retain their full reproductive capacity in ICSI, but raise the concern that, theoretically, ICSI could transmit infectious material as well.