Construction of lentiviral vector carrying human OCT4A gene and establishment of leukemic cell line K562 stably expressing OCT4A.
10.7534/j.issn.1009-2137.2014.06.003
- Author:
Fan-Jing MENG
1
;
Jiang CAO
2
;
Jun ZHOU
1
;
Qing-Yun WU
2
;
Chong CHEN
2
;
Kai-Lin XU
2
;
Author Information
1. Laboratory of Transplantation Immunology, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
2. Laboratory of Transplantation Immunology, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China
- Publication Type:Journal Article
- MeSH:
Cell Cycle;
Cell Proliferation;
Genetic Vectors;
Green Fluorescent Proteins;
Humans;
K562 Cells;
Lentivirus;
Leukemia;
genetics;
pathology;
Octamer Transcription Factor-3;
genetics;
Plasmids;
Real-Time Polymerase Chain Reaction;
Transfection
- From:
Journal of Experimental Hematology
2014;22(6):1514-1520
- CountryChina
- Language:Chinese
-
Abstract:
This study was purposed to construct a lentiviral vector carrying human OCT4A gene and green fluorescent protein (GFP) , and infect the leukemic cell line K562, observe the expression of OCT4A in K562 cells. According to the sequence of OCT4A mRNA which was found in GenBank, the special primer sequences were synthesized. The OCT4A gene was amplified by RT-PCR, and then cloned into the pCR-Blunt vector. The OCT4A DNA fragment was subcloned into the lentiviral vector pLVX-IRES-ZsGreen1 which was restricted by EcoR1 to generate a lentiviral vector pLVX-OCT4A-ZsGreen1. The sequence of the recombinant plasmid was identified by DNA sequencing. Recombinant lentivirus was generated by co-transfection of three-plasmids into 293FT cells using lipofectamine 2000 and transfected into K562 cells. Real-time PCR and Western blot were applied to detect the expression of OCT4A mRNA and protein, CCK-8 and colony formation assay were performed to evaluate the effects of OCT4A on proliferation of K562 cells. The results showed that the recombinant lentiviral vector pLVX-OCT4A-ZsGreen1 was successfully constructed. The virus titers were (1.43 ± 0.25) × 10(8) U/ml. After infection of K562 cells with the lentivirus, the recombinant plasmid could stably up-regulate the expression of OCT4A gene and protein according the real-time PCR and Western blot detection results. CCK-8 and colony formation assay showed that exogenous OCT4A gene could significantly promote cell growth and the colony formation of K562 cells. It is concluded that the recombinant lentiviral vector pLVX-OCT4A- ZsGreen1 carrying human OCT4A gene is successfully constructed; K562 cells which stably up-regulates the expression of OCT4A mRNA are obtained, the results of this study provide fundamental basis for further study on mechanism of OCT4A in human leukemia development.
