Construction of recombinant adenovirus vector carrying human miR-133a and its expression in vitro
- VernacularTitle:人微小RNA-133a(miR-133a)重组腺病毒的构建和鉴定
- Author:
Bin ZHANG
;
Zhixin SHAN
;
Qiuxiong LIN
;
Zhiling ZHOU
;
Chunyu DENG
;
Ailin GUO
;
Yongheng FU
;
Honghong TAN
;
Xiyong YU
- Publication Type:Journal Article
- Keywords:
microRNA;
Vector construction;
Bone marrow mesenchymal stem cells;
Edenovirus
- From:
Chinese Journal of Pathophysiology
2000;0(07):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM:To construct recombinant adenovirus vector carrying human miR-133a and study its expression in human mesenchymal stem cells(hMSCs).METHODS:The PCR product containing miR-133a was amplified from human genomic DNA and inserted into the adenoviral shuttle vector pAdTrack-CMV.Then the recombinant shuttle plasmid linearized by pmeⅠwas cotransformed into competent E.coli.BJ5183 with the adenoviral backbone plasmid pAdEasy-1 to generate the recombinant adenovirus vector rAd-mir-133a.rAd-mir-133a was then packaged and amplified in human embryonic kidney 293(HEK293) cells.The purified rAd-miR-133a was used to infect the hMSCs and the expression of miR-133a was detected by non-quantitative RT-PCR and real-time PCR.RESULTS:The recombinant adenovirus shuttle vector pAdTrack-CMV-miR-133a was constructed and verified by restriction endonuclease analysis and DNA sequence analysis.rAd-miR-133a was successfully packaged and amplified in HEK293 cells.The transcriptions of primary miR-133a and mature miR-133a were over-expressed in the hMSCs infected with rAd-miR-133a.CONCLUSION:The recombinant adenovirus vector carrying human miR-133a is successfully constructed,which lay a foundation for miR-133a function study.
