Construction of double expression retroviral vectors and its effect on phenotype of K562 cells
- VernacularTitle:重组逆转录病毒双表达载体的构建及其对K562细胞增殖活性的影响
- Author:
Jianming ZENG
;
Wenli FENG
;
Xiaozhong WANG
;
Shiqiao ZHAO
;
Weijun BAI
;
Yunping LUO
;
Jianping WEN
;
Zhiguang TU
;
Zongga HUANG
- Publication Type:Journal Article
- Keywords:
chronic myeloid leukemia;
PKR;
antisense RNA;
retroviral vector;
gene clone
- From:Journal of Third Military Medical University
2003;0(21):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct double expression retroviral vectors targeting chronic myeloid leukemia (CML) b3a2-type mRNA and investigate its effect on the phenotype of K562 cells. Methods The eGFP coding sequence was inserted into the retroviral vector pMSCV-neo to construct pMSCV/GFP, then H1-RNA pol III-based transcription cassettes was subcloned into it to form pMSCV/GFP-H1-BCR/ABL40AS. Two control vectors pMSCV/GFP-H1-BCR/ABL40S & pMSCV/GFP-H1-BCR/ABL80AS were constructed in addition. All these constructions were identified by restriction enzyme analysis and DNA sequencing. After that, the recombinant vectors were transferred into retrovirus packaging cell line PT67 by using lipofectamine2000, and G418 were used to select stable virus-producing cell lines. Viral titer was determined by infection of NIH3T3 cells sequentially. The cell-growth curve was assayed, cell apoptosis was checked with Annexin V-PE/7AAD double staining and flow cytometry analysis after 24-hour infection, the PKR phosphorylation was assayed by Western blotting. Results The plasmids were successfully constructed. Four cell lines, named as PT67-MSCV/GFP, PT67-40as, PT67-40s and PT67-80as were gained by G418 selection, and virus titers were 6.2?10~ 5 , 5.6?10~ 5 , 4.6?10~ 5 and 6.0?10~ 5 CFU/ml respectively. PT67-40as suspensions could induce K562 cell apoptosis by (22.54?3.19)%, significantly different from PT67-MSCV/GFP or PT67-40s (P
