An Evaluation of Sperm-mediated Gene Transfer in the Pig
L.C. Bolling, R.S. Pleasant, S.P. Butler, W.H. Velander and F.C. Gwasdauskas
The objectives of this study were to examine the influence of linear DNA on aspects of intracytoplasmic sperm injection (ICSI) using membrane-disrupted spermatozoa and in vitro fertilization (IVF) as a prelude to sperm-mediated gene transfer in the pig. Porcine oocytes were shipped overnight in maturation media at 39 °C in a portable incubator. After 22 h of maturation culture, oocytes were washed in maturation medium without gonadotropins and cultured for an additional 22 h. Cumulus cells were removed and oocytes were divided into four treatment groups: treatment 1 = ICSI using membrane-damaged spermatozoa co-incubated with linear green fluorescent protein (GFP) DNA; treatment 2 = ICSI using membrane damaged spermatozoa; treatment 3 = IVF with frozen-thawed spermatozoa co-incubated with linear GFP DNA prior to IVF; and treatment 4 = IVF with frozen-thawed spermatozoa with no DNA. Although no overall difference in development score was observed following the four different treatments, a treatment difference among cleaved oocytes was observed when comparing only the two ICSI treatments (P < 0.05); development scores were greater in the ICSI treatment in which sperm were not co-incubated with linear GFP DNA prior to injection than when the co-incubation was performed. No differences in development score were observed in the two IVF treatments. The percentage of embryos expressing the GFP transgene on d 6 of culture following fertilization was 7.3% in the ICSI+GFP group and 0% in the other treatment groups. Thus, sperm-mediated gene transfer using ICSI in the pig has been demonstrated, although success rates were low.