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 exp ressing embryos in rhesus macaques (Macaca mulatta, mean = 34.6%; n = 81). Rhodamine-tagged DNA encoding the green fluorescent protein (GFP) gene bind s avidly to spermatozoa. The rhodamine signal, while lost at the egg surfac e during in-vitro fertilization (IVF), is traced by dynamic imaging during ICSI and remains as a brilliant marker on the microinjected spermatozoa wit hin the oocyte cytoplasm. The transgene is expressed in preimplantation emb ryos produced by ICSI, but not IVF, as early as the 4-cell stage with the n umber of expressing cells and the percentage of expressing embryos increasi ng during embryogenesis to the blastocyst stage. The three offspring that r esulted from seven embryo transfers (a set of anatomically normal twins, on e male and one female, stillborn 35 days premature, and a healthy male born at term) demonstrate that primate spermatozoa with exogenously bound DNA r etain their full reproductive capacity in ICSI, but raise the concern that, theoretically, ICSI could transmit infectious material as well.