A Preliminary Study On The Generation Of Transgenic Embryos By Sperm-Mediated Gene Transfer In Cattle

In this study, the factors influencing the transgenic efficiency by sperm mediated gene transfer (SMGT) was investigated, so as to set up the platform for producing transgenic cattle by SMGT.Firstly, the effects of the washing times of bovine sperm from different individuals, the time and concentration of sperm incubating with IFN-EGFP plasmid, as well as the ratio of linear/circular plasmid on bovine transgenic efficiency by IVF-MGT were studied. The bovine sperms from Guangjuan NO.3、NO.6and NO.10were respectively washed one to four times, and then incubated with IFN-EGFP plasmid, the result showed that the sperm motility of NO.3and NO.6was significantly decreased with increasing of washing times (P<0.05), but the sperm motility of NO.10in the group of washing twice was no significant difference with those of washing one or three times (0.69vs0.77and 0.65,P>0.05), moreover, it was significantly higher than that of NO.3and NO.6(0.65vs0.57and0.56, P<0.05) when sperm was washed three times. On the contrary, the efficiency of sperm binding exogenous plasmid was increased with the increasing of sperm washing times, when sperm was washed two or three times, the positive rate of sperm binding exogenous plasmid of NO.10was significantly higher than that of NO.3and NO.6(P<0.05). As the sperms from No.10were respectively incubated with plasmid for different time (10min,30min,60min,90min,120min), the sperm motility of10min group and30min group was not significant difference with the control group (P>0.05) and evidently higher than that of60min,90min,120min groups (P<0.05), but it could obtain more the positive rate of sperm binding exogenous plasmid in30min group than that of10min group (20.98%vs10.62%, P<0.05), and was not significant difference with that of60min,90min,120min groups (P>0.05). Furthermore, the rate of cleavage embryos and blastocyst of30min group were significantly higher than60min group (63.84%vs50.52%;23.52%vs15.77%, P<0.05), but the rate of EGFP-positive cleavage embryos and EGFP-positive blastocyst of30min group and60min group were not significant difference (P>0.05). The effect of the different concentration of sperm incubating with plasmid on IVF-MGT transgenic efficiency were evaluated and found that the rate of positive sperm in75ng/μl plasmid group was significantly higher than the other concentration groups (P<0.05), however, the rate of EGFP-positive cleavage embryos and EGFP-positive blastocyst in50ng/μl plasmid group after IVF-MGT were significantly higher than that of25ng/μl and100ng/μl group respectively (38.66%vs26.94%,23.60%;19.05%vs7.14%,5.56%, P<0.05), and there was no significant difference comparing with75ng/μl group (P>0.05). While the sperms were respectively incubated with different ratio of linear/circular plasmid (0:1,1:0,1:1), there were no significant difference in the positive rate of sperm binding exogenous plasmid, embryonic development and transgenic efficiency among groups of the different ratio of linear/circular plasmid (P>0.05).Secondly, the effects of bovine sperm pretreating method and incubation concentration of IFN-EGFP plasmid on ICSI-MGT transgenic efficiency were explored. The results showed that sperm treated with freezen-thawed had a extremely higher rate in binding exogenous plasmid than that of sperm treated by simple co-incubation or NaOH groups (48.47%vs31.09%,37.19%, P<0.05), and more embryos could develop to the blastocyst in the freezen-thawed group after ISCI-MGT comparing with NaOH group (36.15%vs32.11%, P<0.05), moreover, the rate of EGFP-positive blastocyst in the freezen-thawed group was markedly higher than that of simple incubation group (28.29%vs10.28%, P<0.05). For different concentrations of plasmid (5ng/μl,25ng/μl,50ng/μl,75ng/μl,100ng/μl) incubating with sperm, although there was a significantly higher positive rate of sperm binding exogenous plasmid when sperm incubated with75ng/μl or100ng/μl of plasmid than that of5ng/μl,25ng/μl and50ng/μl groups (51.07%,51.19%vs17.87%,31.51%,46.14, P<0.05);the rate of cleavage ICSI embryos of75ng/μl group was significantly lower than other groups (P<0.05), and the rate of EGFP-positive cleavage ICSI embryos of5ng/μl group was significantly lower than other experimental groups (P<0.05). The blastocyst rate of50ng/μl group was significantly higher than75ng/μl group; and blastocysts positive rate of50ng/μl group was significantly higher than other groups (P<0.05).These results suggest that (1) The different individual and washing times of bovine sperm have a profound influence on sperm motility and efficiency of binding exogenous IFN-EGFP plasmid.(2) The time and concentration of sperm incubating with IFN-EGFP plasmid impact on bovine transgenic efficiency by IVF-MGT, and50ng/μl of plasmid and incubation with sperm for30min is suitable for producing bovine transgenic embryos by IVF-MGT.(3) The ratio of linear/circular IFN-EGFP plasmid has no significant effect on bovine transgenic efficiency by IVF-MGT.(4) Sperm treatment and IFN-EGFP plasmid concentration have an influence on ICSI-MGT transgenic embryonic development,and the method of sperm pretreated with freezen-thawed and incubated with50ng/μl plasmid can improve the efficiency of producing bovine transgenic embryos.