Construction and identification of a short hairpin RNA expression vector targeting the Cbl-b gene
10.3760/cma.j.issn.0412-4030.2015.03.018
- VernacularTitle:Cbl-b基因shRNA干扰载体的构建及鉴定
- Author:
Bin HU
;
Nana NI
;
Yalin LYU
;
Hao CHEN
;
Yi LIU
;
Jianfang SUN
- Publication Type:Journal Article
- Keywords:
Ubiquitin-protein ligases;
RNA,small interfering;
Melanoma;
Genes,Cbl-b
- From:
Chinese Journal of Dermatology
2015;48(3):204-207
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a eukaryotic expression plasmid vector encoding Cbl-b gene-specific short hairpin RNAs (shRNAs),and to evaluate its interference effect,so as to lay a foundation for further study on the role of Cbl-b in the immunotherapy of malignant melanoma.Methods According to the sequence of Cbl-b cDNA,4 pairs of shRNAs targeting the Cbl-b gene were designed and synthesized,and then inserted into the plasmid PGPU6/GFP/Neo to construct recombinant plasmids.After identification by DNA sequencing,the 4 shRNA expression vectors were cotransfected into 293T cells with the Cbl-b gene eukarytic expresson plasmid,respectively.The knockdown efficiency of these shRNA expression plasmids on Cbl-b expression was evaluated by real-time (RT) fluorescence-based quantitative PCR and Western blot at 48 hours aftert transfection.Results Sequencing analysis revealed that all the 4 pairs of shRNAs were successfully inserted into the eukarytic expression vector PGPU6/GFP/Neo.As RT-PCR and Western blot showed,all the 4 shRNA-expressing vectors could downregulate Cbl-b expession,and the NO.1 shRNA-expressing vector displayed the strongest interference effect(P ＜ 0.05).Conclusions A eukaryotic expression plasmid vector was successfully constructed for Cbl-b gene-specific shRNAs,and the most effective shRNA was selected in this study.
