Construction and identification of a short hairpin RNA expression vector targeting the Cbl-b gene / 中华皮肤科杂志
Chinese Journal of Dermatology
;
(12): 204-207, 2015.
Article
in Chinese
| WPRIM
| ID: wpr-468678
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Type of study:
Diagnostic study
Language:
Chinese
Journal:
Chinese Journal of Dermatology
Year:
2015
Type:
Article
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