荧光定量PCR精确检测人胚胎干细胞线粒体DNA拷贝数方法的建立.docVIP

  荧光定量 PCR 精确检测人胚胎干细胞线粒 体 DNA 拷贝数方法的建立# 孙懿1,2，曾思聪1,2，卢光琇1,2，林戈1,2** 5 10 15 20 25 30 35 （1. 中南大学生殖与干细胞研究所，湖南长沙 410078； 2. 国家干细胞工程研究中心，湖南长沙 410078） 摘要：建立一种精确定量人胚胎干细胞线粒体 DNA 拷贝数的方法。构建包含线粒体 DNA ND1 和核单拷贝基因β-globin 基因序列的重组质粒作为标准品；收集无饲养层培养体系下 人胚胎干细胞 DNA 样本，结合 2 个单独的 Taqman 探针实时荧光定量 PCR 对待测样本中线 粒体 ND1 和核β-globin 基因分别进行定量，从而对人胚胎干细胞线粒体 DNA 的含量进行 了精确定量。结果提示：人胚胎干细胞线粒体 DNA 的平均拷贝数/细胞为(1321±228)。表明 该技术可对人胚胎干细胞线粒体 DNA 拷贝数进行准确的测定,为研究培养条件对人胚胎干 细胞线粒体 DNA 拷贝数的影响, 并优化体外培养条件奠定基础。 关键词：干细胞工程学；人胚胎干细胞；线粒体 DNA； DNA 拷贝数 中图分类号：Q291, Q813. 1 Method Establishment for precise determination of mitochondrial DNA copy in human embryonic stem cells with real-time PCR SUN Yi1,2, Zeng Sicong1,2, LU Guangxiu1,2, LIN Ge1,2 (1. Institute of Reproductive and Stem Cell Engineering , Central South University , Changsha, Hu’nan 410078 , China; 2. National Research and Engineering Center of Human Stem Cells, Changsha, Hu’nan 410078, China) Abstract: In this paper, a precise assay that determines mtDNA levels in human embryonic stem cells was developed using a real-time PCR-based procedure. Human embryonic stem cells were cultured on feeder free system. Quantification was performed by reference to a single recombinant plasmid standard containing a copy of each target DNA sequence including mitochondrial ND1 gene and single copy β-globin gene of nuclear. Copy number of mtDNA was determined by amplifying a short region of the ND1 gene, and nuclear DNA content was determined by amplification of a segment of the single copy β-globin gene separately. The copy number of mtDNA per diploid nuclear genome in human embryonic stem cells was 1321±228. This study shows that PCR-based assay enables accurate determination of mtDNA relative to nuclear DNA, which lays the foundation for the study of culture conditions effect on mitochondrial DNA copy number in human embryonic stem cell, and for the optimized culture conditions in vitro. Key words: Stem cell engineering; human embryonic stem cell; mitochondrial DNA; DNA C