Construction and identification of RNAi lentiviral vectors targeting human midkine gene
10.13315/j.cnki.cjcep.2014.04.001
- VernacularTitle:人Midkine基因RNAi慢病毒载体的构建与鉴定
- Author:
Qingling WANG
;
Peng ZHANG
- Publication Type:Journal Article
- Keywords:
Midkine;
RNA interference;
lentivirus vector
- From:
Chinese Journal of Clinical and Experimental Pathology
2014;(4):355-359
- CountryChina
- Language:Chinese
-
Abstract:
Purpose To construct a lentiviral ventor-mediated RNA interference of human midkine ( MK) gene and to provide the ba-sis for further experiment in vivo and in vitro. Methods Four RNAi sequences targeting human MK gene were designed, and cloned into the lentiviral vector to construct lentiviral vectors:GV115-MK-1, GV115-MK-2, GV115-MK-3 and GV115-MK-4. After transfec-tion into competent E. Coli bacteria, the candidate clones were identified by PCR and DNA sequencing. The titer of lentiviruses was determined after 293 T cells were co-transfected with GV115-MK, pHelper 1. 0 and pHelper 2. 0. The four kinds of recombinant lenti-viruses were used to infect human breast cancer cell MDA-MB-231, and the expression levels of MK mRNA were detected by real-time PCR. Results PCR analysis and DNA sequencing confirmed that the four inserted MK RNAi sequences were corrected. Strong green fluorescence was observed in the 293 T cells under the fluorescent microscope after co-transfection. The titer of the concentrated virus was 6 × 108 , 5 × 108 , 5 × 108 and 6 × 108 TU/ml, respectively. The MK expression in MDA-MB-231 cells infected with lentiviral vec-tors GV115-MK-1 was decreased by 87. 2% (P<0. 05). Conclusion Lentiviral RNAi vector for MK gene is successfully construc-ted, and it could effectively inhibit the expression of MK gene in MDA-MB-231 cells in vitro.
