Bull Sperm Uptake of Exogenous DNA and Efforts to Obtain Transgenic Embryos
Saksiri Sirisathien, Levent Keskintepe and Benjamin G. Brackett
The use of bull spermatozoa as an alternative, non-invasive gene transfer method for production of transgenic embryos was investigated. Ejaculated, and epididymal spermatozoa were incubated for increasing intervals with a fluorescence labeled plasmid. Ejaculated bull sperm showed no signs of bound plasmid. In contrast, proportions of epididymal spermatozoa that showed signs of bound plasmid at the end of 1, 2, and 4 h incubations were 86%, 98%, and 98%, respectively, and proportions of epididymal spermatozoa populations retaining bound plasmid after DNase I treatment were 16%, 44%, and 40%, respectively. No transgene expression was observed in bovine blastocysts resulting from in vitro fertilization with epididymal spermatozoa preincubated with plasmid DNA for 2 h. However, 30% of those blastocysts were positive for the presence of transgene after PCR analyses. Intracytoplasmic spermatozoa injection (ICSI) of oocytes with DNA-treated epididymal spermatozoa, freeze-dried, and membrane disrupted spermatozoa was also carried out. No transgene expression was observed in blastocysts that developed in vitro after ICSI with DNA-treated epididymal spermatozoa. Only 3 and 4 embryos (3%) showed evidence of transgene expression after ICSI with membrane disrupted, or freeze-dried spermatozoa that were pre-treated with plasmid DNA. Results of this investigation emphasize the need for further study to develop a repeatable bovine procedure for sperm mediated transgenesis.