不同穿膜肽载体转染对猪精子受精能力的影响

【目的】明确修饰基因组通过穿膜肽载体转染精子的可行性及穿膜肽载体对精子和受精卵的影响,为实现安全有效地大批量制备猪转基因胚胎提供技术支撑。【方法】以水牛Sohlh2基因为目的基因,通过细胞穿膜肽(C105y、MPG和TAT)和慢病毒介导转染猪精子后,采用激光共聚焦扫描显微镜观察转染精子形态特征的变化,利用精子图像分析仪(CASA)检测精子活率、平均曲线运动速度(VCL)、平均直线运动速度(VSL)和前向性(STR)等指标,运用精子顶体染色试剂盒测定猪精子顶体反应,并通过体外受精进一步验证不同载体转染制备生产转Sohlh基因猪胚胎的效果。【结果】转染后的猪精子顶体结构完整,细胞膜未见破裂;不同细胞穿膜肽(C105y、MPG和TAT)的猪精子转染阳性率分别为56.74%、50.44%和43.58%,低于慢病毒的转染阳性率(61.48%),但差异不显著(P>0.05,下同)。经穿膜肽C105y转染24 h,猪精子活率(70.09%)、平均直线运动速度(35.36μm/s)、平均曲线运动速度(42.20μm/s)、前向性(1.03μm/s)与对照组相比差异均不显著;而经穿膜肽MPG和TAT及慢病毒转染后,猪精子的各项指标均呈一定程度的下降趋势。细胞穿膜肽转染猪精子的自发顶体反应率与对照组相比无显著差异,慢病毒转染则呈显著的上升趋势(P<0.05,下同);在诱发顶体反应率方面,慢病毒转染猪精子的诱发顶体反应率较对照组呈显著下降趋势,而不同细胞穿膜肽转染对猪精子诱发顶体反应率也无显著影响。慢病毒和穿膜肽C105y转染猪精子经体外受精获得的受精卵继续培养24 h后其卵裂率为64.82%和65.91%,与对照组受精卵的卵裂率(64.52%)差异不显著;穿膜肽C105y转染组的囊胚率(15.82%)与对照组间无显著差异,而慢病毒转染组的囊胚率(9.11%)显著低于对照组;在转基因胚胎阳性率方面,穿膜肽C105y转染组显著低于慢病毒转染组(8.54%vs 10.38%)。【结论】穿膜肽C105y对猪精子的受精能力及转基因猪胚胎发育影响小,且毒害作用不明显,是一个能高效转导外源基因的安全载体。

Effects of different transmembrane peptide carriers on pig sperm insemination ability

【Objective】To clarify the feasibility of transfection of modified genome into spermatozoa by transmembrane peptide and the effect of transmembrane peptide on spermatozoa and fertilized eggs,so as to provide technical support for the safe and effective mass production of pig transgenic embryos.【Method】With buffalo Sohlh2 gene as the target gene,pig spermatozoa were transfected by cell membrane penetrating peptide(C105y,MPG and TAT)and lentivirus.Morphological characteristics of transfected spermatozoa were observed by laser confocal scanning microscope. Sperm viability,mean curvilinear velocity(VCL),mean linear velocity(VSL)and forward tropism(STR)were detected by sperm image analyzer(CASA),Sperm acrosome staining kit(psa-fitc)was used to detect pig sperm acrosome reaction,and in vitro fertilization was used to further verify the effect of different vector transfection on the production of Sohlh transgenic pig embryos.【Result】The acrosome structure of transfected pig sperm was intact,and the cell membrane was not broken. The positive rates of pig sperm transfection with different cell penetrating peptides(C105y,MPG and TAT)were 56.74%,50.44% and 43.58% respectively,which were lower than that of lentivirus(61.48%),but the difference was not significant(P>0.05,the same below). After transfection with membrane penetrating peptide C105y for 24 hours,the pig sperm viability(70.09%)and the average linear velocity(35.36 μm/s),average curve velocity(42.20 μm/s),forward directivity(1.03 μm/s),compared with the control group,the difference was not significant. After transfection with membrane penetrating peptides MPG,TAT and lentivirus,the indexes of pig sperm showed a downward trend to some extent. There was no significant difference in the incidence of spontaneous acrosome reaction in pig spermatozoa transfected with cell penetrating peptide compared with the control group,but there was a significant upward trend in lentivirus transfection(P<0.05,the same below). In the aspect of inducing acrosome reaction rate,the rate of inducing acrosome reaction in pig sperm transfected with lentivirus was significantly lower than that in the control group,while the rate of inducing acrosome reaction in pig sperm transfected with different cell penetrating peptides had no significant effect.The cleavage rates of the fertilized eggs obtained from porcine sperm transfected with lentivirus and transmembrane peptide C105y after in vitro fertilization were 64.82% and 65.91% after 24 hours of culture,which were not significantly different from those of the control group(64.52%). There was no significant difference in the blastocyst rate(15.8%)between the transfected group and the control group,but the blastocyst rate(9.11%)in the lentivirus transfected group was significantly lower than that in the control group. In terms of the positive rate of transgenic embryos,the membrane penetrating peptide C105y transfection group was significantly lower than that of the lentivirus transfection group(8.54%vs 10.38%).【 Conclusion 】Membrane penetrating peptide C105y has little effect on the fertilization ability of pig sperm and the development of transgenic pig embryos,and its toxic effect is not obvious. It is a safe vector for efficient transduction of foreign genes.