小鼠xist探针载体的构建及鉴定  

Construction and identification of mouse xist probe vector

作　　者：白光宇[1] 申景岭[1] 孙瑞珍[1] 雷蕾[1] 

机构地区：[1]哈尔滨医科大学组织胚胎学教研室,哈尔滨150081

出　　处：《解剖科学进展》2015年第4期364-366,共3页Progress of Anatomical Sciences

摘　　要：目的构建xist探针载体并检验其合成出的探针效果。方法 B6D2F1 BAC(R3026)经PCR扩增获得xist基因,与双酶切的pSPT19质粒连接,经转化及测序鉴定pSPT19-xist载体构建成功。将构建好的pSPT19-xist载体线性化,并体外转录为RNA探针,应用荧光原位杂交检测雌性B6D2F1小鼠成纤维细胞内的xist表达。结果经琼脂糖凝胶电泳,获得目的基因xist条带。基因测序证实所检测重组质粒序列与xist基因序列完全一致。体外雌性小鼠成纤维细胞检测到xist阳性表达。结论成功构建xist基因pSPT19-xist重组质粒和RNA探针,并在成纤维细胞阳性表达。Objective To construct xist probe vector and identify xist protein expression in fibroblasts. Methods The DNA of xist was amplified using polymerase chain reaction（PCR） from B6D2F1 BAC（R3026）, and was double digested by enzymes to be ligated with pSPT19. The recombinant vector pSPT19-xist was identified by transformation and DNA sequencing. The recombinant vector pSPT19-xist was lined and transcribed to obtain the probe in vitro, and then expression of xist in the female mouse B6D2F1 fibroblast was detected by fluorescence in situ hybridization. Results The target gene xist was obtained through electrophoresis analysis, the sequence of recombinant vector was same as xist by DNA sequencing. The expression of RNA xist was detected in the female mouse fibroblasts. Conclusion The recombinant pSPT19-xist vector was successfully constructed and the protein was positively expressed in fibroblasts.

关 键 词：B6D2F1鼠 载体构建 RNA探针 成纤维细胞 

分 类 号：Q781[生物学—分子生物学]