逆转录病毒转染胚盘细胞制备嵌合体鸡胚的研究

Studies on Production of Chimeraic Chicken Embryos via Transfected Blastoderm Cells

【目的】通过重组逆转录病毒体内感染源于第X期胚盘细胞的方法获得嵌合体鸡。【方法】将扩增的增强型绿色荧光蛋白(EGFP)基因的开放阅读框插入逆转录病毒表达载体pour-pBabe中获得重组逆转录表达载体;采用磷酸钙转染法将其与包装载体pVSVG共转入29310A1包装细胞中进行病毒包装,得到病毒原液经超速离心浓缩200倍后注入种蛋(第X期)胚盘下腔,孵化种蛋。提取孵化5.5天的鸡胚基因组DNA,进行PCR检测和Southern杂交检测。【结果】构建含EGFP完整开放阅读框的逆转录病毒表达载体pour-pBabe-EGFP,提取孵化5.5d鸡胚基因组DNA,经PCR、Southern杂交检测,嵌合体阳性率分别为75%和66.7%。【结论】通过用重组逆转录病毒体内感染鸡胚盘细胞的方法可获得嵌合体鸡。

【Objective】 Chicken chimeras were produced using chicken stage X blastoderm cells, which were infected by recombinant retrovirus in vivo. 【Method】 The recombinant expression vector pour-pBabe-EGFP was established by connecting expression vector and EGFP gene. The recombinant vector and packaging vector pVSVG were transfected into 293 10A1 cells together using calcium phosphate transfection to produce retrovirus. The concentrated retroviruses （200×） were injected into the central part of the subgerminal cavity to transfect the PGCs in eggs （not incubated）. These eggs were incubated and then selected at random when they were hatched at 5.5 days. The genomic DNA were extracted and identified by polymerase chain reaction （PCR） and Southern blot analysis. 【Result】 The recombinant expression vector pour-pBabe-EGFP, which contained the full ORF, was established Successfully. The positive ratio was 75% by PCR analysis and 66.7% by Southern blot analysis. 【Conclusion】 The chicken chimeras were obtained successfully. Chicken chimeras can be produced using chicken blastoderm cells infected by recombinant retrovirus in vivo.