Construction of human DNMT3A lentiviral vector and establishment of stale MDA-MB-231 cell line

ZHANG Huibian; WANG Yuan; YU Lin

doi:10.3969/j.issn.1002-266X.2017.42.002

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.