鸡cvh启动子调控的绿色荧光蛋白报告载体的构建及鉴定

Construction and Identification of gfp Reporter Vectors Driven by Chicken cvh Gene Promoters

探索建立一种用绿色荧光蛋白对鸡原始生殖细胞进行标记分离的方法。克隆并测序鸡原始生殖细胞特异表达基因cvh1.6kb启动子序列。序列分析表明它含有类似于TATAbox和CAATbox的元件,在其远端上游区域还有多个GC富含区。将cvh基因启动子亚克隆到绿色荧光蛋白表达载体pegfp-1的多克隆位点,成功构建表达载体pcvh-egfp。重组质粒在脂质体LipofectamineTM2000介导下分别转染鸡Ⅹ期胚盘细胞和鸡胚成纤维细胞,并于转染后12h在荧光显微镜下观察转染效果。实验结果显示:在胚盘细胞转染后12h可观测到绿色荧光表达,转染后24h荧光细胞增多,细胞形态均呈圆形、体积较大;在鸡胚成纤维细胞未检测到gfp表达。实验结果说明,由cvh基因启动子控制下的gfp可在Ⅹ期胚盘细胞特异表达,为利用流式分离技术PGCs标记分离、纯化研究奠定基础。

In chicken, the distribution of cvh transcripts is restricted to the germ cell lineage, making it a useful indicator of PGCs. In order to label live PGCs, we cloned and characterized the chickenl.6kb cvh gene promoter regions. TATA box and CAAT box-like elements were found in the sequence, and GC domains were rich in the far upsteam of the promoter. The gfp reporter vectors （pcvh-eg]：p） driven by cvh gene promoters was constructed. The blastodermal cell and CEF cell were transfected respectively by Lipofectamine^TM2000 with pcvh-egfp for transient expression. The gfp-labeled positive blastodermal cells were first observed 12 h post-transfection and increased with the post-transfection time （in 24 h）. Gfp-labeled and non-labeled cells from X blastoderm were large and round with the same characters of morphology. The gfp protein was not expressed in CEF cell .Our results suggested that the cvh promoter regions have specific activity in stage X blastoderm. It is possible to develop a method for labeling chicken PGCs with gfp and subsequently to sort viable gfp-positive PGC from somatic cell by flow cytometry. This should be extremely useful for applications of PGC transplantation.