人类成肌发育候选基因ASB12 RNAi稳定细胞系的构建与鉴定

摘要

目的:利用siRNA表达载体构建抑制人类成肌发育候选基因ASB12表达的pSUPER RNAi载体(pSU-PER-ASB12),筛选构建C2C12-pSUPER-ASB12稳定表达细胞系.方法:化学合成一对编码短发夹RNA序列的、靶向成肌发育候选基因ASB12的寡核苷酸链60个碱基,退火,克隆到经BglⅡ、Xho Ⅰ双酶切的pSUPER质粒上,构建重组RNAi质粒(pSUPER-ASB12).通过酶切鉴定及测序分析检测构建效果.将正确构建的质粒转染小鼠骨骼肌细胞C2C12,通过G418筛选,免疫荧光检测,RT-PCR分析,建立稳定表达pSUPER-ASB12的细胞系C2C12-pSUPER-ASB12.结果:pSUPER-ASB12载体经酶切鉴定及测序分析,结果表明60个碱基成功插入到预计位点,并且序列完全一致.C2C 12-pSUPER-ASB 12稳定表达细胞系可表达绿色荧光蛋白,RT-PCR检测结果显示C2C12-pSUPER-ASB12细胞中ASB12表达量明显降低.结论:靶向ASB12的pSUPER RNAi载体和C2C12-pSUPER-ASB12稳定表达细胞系构建成功,为进一步从分子水平探讨ASB12在成肌发育中的功能奠定了基础.%To construct a pSUPER RNAi vector that can inhibit human skeleton muscle developmental candidate gene ASB12 gene expression and establish a stable C2C12-pSUPER-ASB12 cell line. Methods; A pair of 60 ntoligonucleotides coding for short hairpin RNA and targeting human skeleton muscle developmental candidate gene ASB12 were chemically synthesized and annealed and inserted into pSUPER plasmids which was digested with Bgl II and Xho I to construct the recombinant pSUPER RNAi plasmid (pSUPER-ASB12). Recombinant pSUPER-ASB12 plasmid was i-dentified by enzyme digestion and sequencing analysis. The C2C12 cells were transfected with the recombinant plasmid, and screened culture by G418 to "establish the stable C2C12-pSUPER-ASB12 cell line. RT-PCR was used to determine the expression of the ASB12. Results: The result of enzyme digestion and sequencing analysis demonstrated that 60 nt oligonucleotides had been inserted successfully into the vector. The expression of ASB12 was detected by immunofluores-cence and RT-PCR. The C2C12-pSUPER-ASB12 cell line in which the expression of ASB12 is inhabited stably was established successfully. Conclusion: The pSUPER RNAi vector targeting human skeleton muscle developmental candidate gene ASB12 and the stable C2C12-pSUPER-ASB12 cell line were successfully constructed, which established a foundation for the study of the molecular function of ASB12.