山东医药2017，Vol.57Issue(42)：5-8,4.DOI:10.3969/j.issn.1002-266X.2017.42.002
人DNMT3A基因慢病毒载体构建及其乳腺癌细胞MDA-MB-231稳定表达株的建立
Construction of human DNMT3A lentiviral vector and establishment of stale MDA-MB-231 cell line
摘要
Abstract
Objective To construct elentiviral vector of human DNA methyltransferase 3A(DNMT3A) and to es-tablish the stable breast caner MDA-MB-231 cell line.Methods The total RNA was extracted from MDA-MB-231cells and cDNA containing DNMT3A was inverse transcribed. DNMT3A fragment containing EcoRⅠand BamHⅠsites was am-plified by PCR and was inserted into pLVX-IRES-Hyg lentiviral vector. Then the recombinant expression vector pLVX-FLAG-DNMT3A was co-transfected into 293T cell line with packaging plasmids,and the culture supermatant containing the lentiviral particles was collected to infect MDA-MB-231 cells. After 48 h,1 μg/μpuromycin was used to screen the infec-ted cells,so as to obtain a cell line with stable expression. The protein and mRNA expression levels of DNMT3A were de-tected by Western blotting and PCR.Results Bacteria colonies PCR,double restriction enzyme digestion,plasmid PCR identification,and DNA sequencing demonstrated the lentiviral vector pLVX-FLAG-DNMT3A was constructed. MDA-MB-231 cells were infected by the lentivirus,and the protein and mRNA expression of DNMT3A was significantly up-regulated following screening with puromycin.Conclusions The DNMT3A gene lentiviral vector pLVX-FLAG-DNMT3A is con-structed successfully. The cell line stably expressing DNMT3A is screened in MDA-MB-231 cells,which provides an in vitro model for further study of molecular function and mechanism of DNMT3A in human breast cancer.关键词
DNMT3A基因/慢病毒载体/MDA-MB-231细胞/乳腺癌Key words
DNMT3A gene/lentiviral vector/MDA-MB-231 cells/breast carcinoma分类
生物科学引用本文复制引用
张慧变,王媛,于林..人DNMT3A基因慢病毒载体构建及其乳腺癌细胞MDA-MB-231稳定表达株的建立[J].山东医药,2017,57(42):5-8,4.基金项目
国家自然科学基金资助项目(81672592,81202102). （81672592,81202102）
