慢病毒介导EGFP在小鼠胚胎的体外表达  

Expression of EGFP in Mouse Embryos Mediated by Lentivirus

作　　者：许诺[1] 贾俊龙[1] 王维[1] 朱玉博[1] 王雪晗[1] 白文林[1] 罗光彬[1] 

机构地区：[1]沈阳农业大学畜牧兽医学院,沈阳110161

出　　处：《沈阳农业大学学报》2016年第5期559-566,共8页Journal of Shenyang Agricultural University

基　　金：农业部转基因生物新品种培育重大专项基金项目(H022020060420)

摘　　要：为探讨3种感染方法对不同发育阶段胚胎中EGFP基因表达影响,利用透明带下注射法、胞质内注射法及透明带切口与慢病毒共培养法感染1-细胞期小鼠胚胎,分别提取以上3种方法感染慢病毒的不同发育阶段胚胎的EGPF基因,并以其为模板,扩增出630bp的EGPF基因片段。结果表明:慢病毒转染后在不同发育阶段的胚胎中,均扩增出EGFP目的片段(630bp)。当透明带下注射病毒时,90p L注射组(C组)和60p L注射组(B组)的EGFP基因阳性率显著优于30p L注射组(A组)(p<0.05)。当胞质注射病毒时,C组显著优于其他组(p<0.05),但胚胎发育的后期A组和B组差异不显著(p>0.05)。透明带做切口的胚胎与含不同浓度慢病毒共培养时,从1-细胞期到4-细胞期胚胎为止组间差异不显著(p>0.05),而胚胎发育的后期2×103cfu·μL^(-1)组和3×103cfu·μL^(-1)组均极显著优于1×103cfu·μL^(-1)组(p<0.01),但2×103cfu·μL^(-1)组和3×103cfu·μL^(-1)组间差异不显著(p>0.05)。透明带下注射慢病毒与胞质内注射慢病毒时,注射90p L(病毒滴度3×103cfu·μL^(-1))组的EGFP基因体外表达效果最好;透明带做切口的胚胎与含不同浓度慢病毒共培养时,在3×103cfu·μL^(-1)滴度培养液中培养的EGFP基因体外表达效果最好。In this research, transparent injection, cytoplasm injection and transparent in c is ion with lentivirus co -cu l tu r e method were used to infect 1 -cell stage mouse embryos, and to explore the different developmental stages of embryos infected with EGFP gene expression in the three methods. EGPF gene was extracted from the above three methods of lentivirus infect ion in different developmental stages of embryos. With EGFP as a template, a 630bp segment of EGFP gene was amplified. Different developmental stages of embryos were transfected by lentivirus, and the ampl ified EGFP fragment （630bp） were obtained. When the virus was injected below transparent film, the EGFP gene positive rate of 90pL injection group （group C） and 60pL injection group （group B） was significantly better than that of 30pL inject ion group （group A） （p〈0.05） . When the virus was injected to the cytoplasm, group C was significantly better than the other group （p 〈 0 .0 5 ） , but later in group A and B difference o f embryonic development was not significant （p〉0.05）. When the embryos with transparent incision were cultured with different concentrations of lentivirus, from 1-cell stage to 4-cell stage embryos among the groups were not significantly differnent （p 〉0.05） , while later the 2 ×10^3cfu· μL-1 group and 3 × 10^3c fu·μ L-1 group were significantly better than 1×10^3cfu · μL-1 group ^ 〈 0 .0 1 ） , but with no difference between 2×10^3c fu·μL-1 group and 3×10^3c fu ·μL-1 group （p〉0.05） . Transparent film or cytoplasm inject ion with the lentivirus, 90pL （viral titer of 3×10^3cfu· μL-1） inject ion group gave the best effect expression in vitro of EGFP gene; the embryos with transparent incision were cultured with different concentrations of lentivirus, 3×10^3cfu·μL -1 titer group showed the best effect expression in vitro of EGFP gene.

关 键 词：慢病毒 胚胎 EGFP 体外表达 

分 类 号：Q78[生物学—分子生物学]