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人类成肌发育候选基因ASB12 RNAi稳定细胞系的构建与鉴定
引用本文:文斗斗,周军媚,廖四芳,宋文,胡维新,吴秀山,王跃群. 人类成肌发育候选基因ASB12 RNAi稳定细胞系的构建与鉴定[J]. 激光生物学报, 2012, 21(4): 335-339
作者姓名:文斗斗  周军媚  廖四芳  宋文  胡维新  吴秀山  王跃群
作者单位:1. 中南大学生物科学与技术学院,湖南长沙,410000
2. 湖南师范大学心脏发育研究中心发育生物学教育部重点实验室,湖南长沙410006;南华大学药学与生命科学学院,湖南衡阳421001
3. 湖南师范大学心脏发育研究中心发育生物学教育部重点实验室,湖南长沙,410006
基金项目:湖南省自然科学基金资助项目,国家自然科学基金资助项目
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、XhoⅠ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。C2C12-pSUPER-ASB12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。

关 键 词:成肌发育  ASB12  pSUPER  RNAi

Construction and Identification of the Stable Cell Line C2C12-pSUPER-ASB12 Expressing siRNA for Human Skeleton Muscle Developmental Candidate Gene ASB12
WEN Doudou , ZHOU Junmei , LIAO Sifang , SONG Wen , HU Weixin , WU Xueshan , WANG Yuequn. Construction and Identification of the Stable Cell Line C2C12-pSUPER-ASB12 Expressing siRNA for Human Skeleton Muscle Developmental Candidate Gene ASB12[J]. ACTA Laser Biology Sinica, 2012, 21(4): 335-339
Authors:WEN Doudou    ZHOU Junmei    LIAO Sifang    SONG Wen    HU Weixin    WU Xueshan    WANG Yuequn
Affiliation:2. (1. College of Life Sciences and Technology, Central South University, Changsha 410000, Hunan, China; 2. The Center for Heart Development, Key Lab of MOE of Developmental Biology, Hunan Normal University, Changsha 410006, Hunan, China; 3. College of Pharmacy and Life Science, University of South China, Hengyang 421001, Hunan, China)
Abstract:Objective:To construct a pSUPER RNAi vector that can inhibit human skeleton muscle developmental can- didate gene ASB12 gene expression and establish a stable C2C12-pSUPER-ASB12 cell line. Methods: A pair of 60 nt oligonucleotides 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 ol- igonucleotides 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 es- tablished 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 founda- tion for the study of the molecular function of ASB12.
Keywords:skeleton muscle development  ASB12  pSUPER  RNAi
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