Construction and Identification of pSIREN-HIF-1α/shRNA Expression Vector
- VernacularTitle:pSIREN-HIF-1α/shRNA表达载体的构建与鉴定
- Author:
Liufang ZHAO
;
Shuping ZHANG
;
Xiaojiang LI
;
Yanxin REN
;
Kang ZHANG
;
Bo YU
;
Jing MA
;
Jun SUI
- Publication Type:Journal Article
- Keywords:
RNA interference;
Nasopharyngeal darcinoma;
Plasmids;
HIF-1α;
shRNA
- From:
Journal of Kunming Medical University
2013;(10):58-61,72
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct and identity pSIREN-HIF-1α/shRNA expression vector in order to make foundation of gene therapy for further exploration of RNA interference to nasopharyngeal darcinoma. Methods According to HIF-1αcDNA gene sequence in the gene bank (NM_001530/NM_181054), a pair of 60 nt oligonucleotides each containing the sites of restriction endonuclease at both ends,were designed and synthesized by Reynolds design principles. Oligonucleotides were annealed and ligated with linedrized RNAi-Ready pSIREN-RetroQ-ZsGreen.Transfected into JM109, the recombinants were finally sequenced and identified by 1%agarose gel electrophoresis. Results The size of the target gene fragment amplified by PCR was 470 bp and in accordance with the expected result.pSIREN-HIF-1α was successfully constructed and identitfied by 1%agarose gel electrophoresis.Sequence analysis of inserted fragment revealed the same sequence as synthesized shRNA Oligonucleotides. Conclusion pSIREN-HIF-1α /shRNA expression vector has been successfully constructed, and can make the foundation of research using liposome packaging transfectiing nasopharyngeal darcinoma cell for the next step .
