Sperm mediated gene transfer (SMGT) is an interesting tool for animal transgenesis, consisting of the use of sperm cells as a vector for transmitting exogenous DNA into eggs at the moment of fertilization. A degradation of sperm membrane followed by incubation with DNA and fertilization using intracytoplasmic sperm injection (ICSI) proved to be efficient in mice (Perry et al. 1999 Science 284, 1180-1183; Moreira et al. 2004 Biol. Reprod. 71, 1943-1947) and rats (Kato et al. 2004 Mol. Reprod. Dev. 69, 153-158). In this study, we evaluated the effect of the sperm treatment (a quick freezing-thawing process for disrupting sperm membranes) on further transgenic expression and the embryo development of injected porcine oocytes. Ejaculated sperm cells from five fertile mature boars were used as vectors for transferring plasmid DNA (GFP: green fluorescent protein) into matured porcine in vitro oocytes by ICSI. Semen was recovered and immediately diluted 1:10 in SFM (swine fertilize medium) at 37�C and later centrifuged (800g, 10 min, 25�C), discarding the seminal plasma to avoid a detrimental effect on DNA binding to cells without further preparation (control), or after they had been subjected to membrane disruption by a quick freeze-thawing process (FT). Linealized plasmid DNA (5.4 kb) was added (1 � 108 sperm/mL + 5 mg DNA/mL) and incubated at 16�C for 30 min. 5 min before ICSI, sperm were pre-warmed at 37�C. Denuded oocytes were washed twice in DPBS medium supplemented with 10% FCS (fetal calf serum) and transferred to ICSI drops. Injected oocytes were kept in TALP medium (Rath et al. 1999 J. Anim. Sci. 77, 3346-3352) for 18 h, and then transferred to NCSU-23 medium for further embryo culture. Embryos were examined for cleavage rate at 48 h following injection, and for embryo development at 144 h. GFP expression in embryos was examined under fluorescent light using a fluorescence inverted microscope. In this preliminary study, 105 and 101 oocytes were injected for control and FT groups, respectively. The cleavage rate was similar between groups (control: 49/105 (46.7%) vs. FT: 39/101 (38.6%); P = 0.25). However, the blastocyst formation rate was lower in the FT than in control group (control: 13/105 (12.4%) vs. FT: 4/101 (4.0%); P = 0.03). In relation to transgenic expression, the FT group showed a significantly higher number of transgenic embryos (control: 50.98 % vs. FT: 75.51%; P = 0.01). These results confirmed that treatment of the sperm prior to the ICSI could affect the efficiency of the production of transgenic embryos. The disruption of sperm membrane increases the DNA binding and the possibilities of carrying the DNA into the oocyte, and freezing-thawing technique is simple and effective to achieve this purpose. However, the freezing process could damage the sperm nucleus structure and decrease the viability of the embryo produced. Nevertheless, the efficiency of the transgenesis expression was very high (more than 75% of the embryos were obtained), and it should be a useful tool to produce transgenic pigs.
This work was supported by AGL2003-03144.
Synthesis, DNA-binding and antiproliferative activity of N-(Nitrogen heterocyclic) norcantharidin acylamide acid
