期刊论文

Construction and Identification of MGC803 Cell Transfacted by RORα Gene

中文

中南医学科学杂志

2095-1116

2016

44

1

5-10

10.15972/j.cnki.43-1509/r.2016.01.002

81374013，81102854:国家自然科学基金 09K074:湖南省高等学校创新平台开放基金 B2015-182:湖南省卫生计生委科研项目

本校为第一机构

目的构建维甲酸相关孤核受体α(RORα)基因的真核表达载体,并建立稳定高表达 RORα的人胃癌MGC803细胞,为研究 RORα的功能奠定基础。方法根据 RORα基因的 cDNA 全序列,设计一对带有限制性酶切位点的引物。从人胃癌 MGC803细胞中提取 mRNA 作为模板合成 RORα cDNA 第一链,并用上述引物扩增目的基因全序列,然后经双酶切插入到真核表达载体 pcDNA3.1(+),转化大肠杆菌 DH5α；用氨苄青霉素筛选 pcDNA3.1(+)-RORα的阳性菌株,双酶切和测序进行鉴定。用经鉴定的重组质粒 pcDNA3.1(+)-RORα转染 MGC803细胞,G418筛选获得 RORα/MGC803细胞株；Real-time PCR 和 Western blot 检测该细胞株 RORα的表...

Objective To construct the eukaryotic expression vector inserted by RORα gene, and establish human gastric cancer MGC803 cell lines that express higher level RORα gene to lay the foundation for investigating the function of RORα. Methods Based on the complete cDNA sequence of RORα isoform, to design a pair of primers with restriction sites. To extract the total mRNA from human gastric cancer MGC803 cells as a template for synthesize first strand cDNA of RORα, and amplify the complete sequences of target gene by the above primers. Then, aft...