慢病毒载体介导的绿色荧光蛋白转基因标记法研究猪嵌合体制作

目的:通过建立慢病毒载体感染猪胚胎体系实现胚胎标记,进而研究不同发育阶段猪孤雌胚胎之间的嵌合能力,为进一步研究猪早期胚胎发育以及细胞分化奠定基础.方法:首先,通过显微注射的方法把2×109I.U./ml、2×108I.U./ml和2×107I.U./ml三个梯度的表达绿色荧光的慢病毒载体分别注射到猪1-细胞胚胎和2-细胞胚胎的透明带下,进行胚胎的GFP转基因标记,在荧光显微镜下观察比较卵裂率、阳性胚胎率、囊胚率、阳性囊胚率和囊胚细胞数.然后,采用凹窝聚合法对同步发育胚胎在不同阶段(2-细胞,4-细胞,8-细胞)进行嵌合,2-细胞胚胎与不同发育阶段(2-细胞、4-细胞、8-细胞)胚胎进行嵌合以及2-细胞胚胎卵裂球互换制作嵌合体胚胎,发育到囊胚时在荧光显微镜下检测胚胎的嵌合状态.结果:2×109I.U./ml的慢病毒感染猪2-细胞胚胎组中,体外受精和孤雌胚胎感染阳性率( 80.00％、76.36％)和阳性囊胚率(90.74％、89.56％)都显著高于其它滴度组(P＜0.05),另外,慢病毒感染的两种胚胎与对照组对卵裂率、囊胚率和囊胚细胞数三个指标没有显著影响(P＞0.05).2-细胞胚胎之间嵌合囊胚率和2-细胞卵裂球互换嵌合囊胚率( 53.85％、62.50％)显著高于2-细胞胚胎与4-细胞胚胎的嵌合率(18.60％,P＜0.05),在同步发育胚胎中8-细胞胚胎之间的嵌合率(75.00％)高于4-细胞胚胎之间和2-细胞胚胎之间的嵌合率( 65.00％、53.80％).结论:2×109I.U./ml的慢病毒感染2-细胞期胚胎效率最高,另外,慢病毒感染对猪胚胎发育没有明显影响.8-细胞间的嵌合率比较高；发育同步胚胎间的嵌合率高于发育非同步胚胎间的嵌合率.

Study on Generation of Chimeric Porcine Embryos Derived from Lentivirus Mediated Green Fluorescent Protein(GFP) Transgene Embryos

Objective: Through the establishment system of the lentiviral vector infected porcine embryo,studied chimeric ability of porcine parthenogenetic embryos of the different development stages,made a foundation for further studying early embryonic development and cell differentiation.Method: The lentivirus with GFP report gene was injected into the perivitelline space of porcine embryos at 1-cell and 2-cell stage,and the cleavage rate,GFP-positive embryo rate,blastocyst rate,GFP-positive blastocyst rate and cell number of blastocyst were examined in fluorescence microscope.Well of well(WOW) method was employed to make chimeric embryos by aggregating two different embryos at the same developmental stage of 2-cell,4-cell and 8-cell or 2-cell stage embryo with 2-cell,4-cell and 8-cell stage embryos,and blastomere exchanging at 2-cell stage was also used for making chimeric embryo.Then the chimerism of porcine embryos were checked.Result: In groups of 2×109I.U./ml lentivirous injecteion in the perivitelline of the 2-cell stage embryos,the infection rate and GFP-positive blastocyst rate of IVF(80.00%,90.74%) and parthenogenetic embryos(76.36%,89.56%) were significantly higher than other groups(P<0.05),and there is no significant difference in cleavage rate,blastocysts rate,blastocysts cell number compared with the control group.The chimeric rate of embryos derived from aggregating of two 2-cell stage embryos(53.85%) and embryos from blastomere exchanging of 2-cell stage embryos(62.50%) were significantly higher than that of aggregating of 2-cell with 4-cell or 8-cell embryos(18.60%,P<0.05),and the chimeric rate of embryos derived from aggregating of two 8-cell embryos(75.00%) were significantly higher than that of these embryos derived from aggregating of two 4-cell embryos(65.00%)or 2-cell embryos(53.80%).Conclusion: 2×109I.U./ml of lentivirous injection in the perivitelline of two-cell stage embryos shows the highest infection efficiency,in addition,lentivirus infection has no significant effect on developmental of porcine embryos.The chimeric rate of embryos derived from aggregating two 8-cell embryos is higher than the others.The chimerism rate of the synchronized developmental embryos is higher than that of the non-synchronized embryos.