Construction of lentiviral vector for shRNA of MACC1 gene and its transfection into MB-231
10.3969/j.issn.1671-8348.2014.12.022
- VernacularTitle:MACC1基因shRNA 慢病毒表达载体的构建及转染MB-231细胞
- Author:
Jie MIN
;
Gang LV
;
Ting WANG
;
Lin GAN
;
Zhen ZENG
;
Yadong WANG
- Publication Type:Journal Article
- Keywords:
metastasis-associated in colon cancer l;
RNA interference;
lentiviral vector MB-231
- From:
Chongqing Medicine
2014;(12):1474-1475,1479
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a lentiviral vector for RNA interference(RNAi)of MACC1 gene and to detect the best trans-fection condition by transfected into MB-231 cells .Methods The siRNA was designed and converted into cDNA of shRNA (small hair pin RNA) of siRNA for MACC1 gene .The cDNA was synthesized and inserted into pMAGic lentiviral plasmid vector which was linearized by enzyme Age Ⅰ and EcoRⅠ .The recombinant plasmid was transformed into competent E .coli DH5α cells .The positive recombinant colony was selected by ampicillin medium agar and identified by DNA sequencing .The recombinant lentivirus was packaged into mature lentivirus by 293FT cells and used to infect MB-231 cells .To detected the transfection condition of high efficiency of infection and low multiplicity of infection .Results PCR and sequencing verified that the recombinant lentivirus plasmid MACC1-shRNA was successfully constructed .The best transfection condition was MOI=40 by transfected into MB-231 .Conclu-sion The lentiviral RNAi expression vector targeting MACC 1 gene is successfully constructed and it can infect MB-231 cells effi-ciently ,which lays the experimental foundation for the research on the changes of malignant biological activity of tumor cell lines and gene therapy .
