Construction and identification of eukaryotic expression vectors expressing shRNA sections targeting human MCHR2
- VernacularTitle:人MCHR2基因shRNA真核表达载体构建及鉴定
- Author:
Chengfu YUAN
;
Junxia YANG
;
Lili WEI
- Publication Type:Journal Article
- Keywords:
genes,MCHR2;
short hairpin RNA;
eukaryotic expression vector;
cell transfection
- From:
Medical Journal of Chinese People's Liberation Army
1981;0(04):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct eukaryotic expression vectors expressing short hairpin RNA(shRNA)sections targeting human MCHR2 and to observe their effects on MCHR2 gene expression in HEK293 cell line.Methods According to the sequence of human MCHR2 gene,the oligonucleotides of shRNA were designed and synthesized and directionally cloned into plasmid pGenesil-1 with enhancing green fluorescence protein(EGFP)gene and Kan gene.The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing.The recombinant vectors were transfected into HEK293 cell line by LipofectamineTM2000,the effects on MCHR2 at mRNA and protein levels were observed.Results Four shRNA expressing recombinants and the corresponding negative control vector were constructed and transfected into HEK293 cell successfully.MCHR2 transcript was reduced by about 45.8%-66.4%,the protein of MCHR2 was reduced by about 44.2%-81.0% in four transfectants respectively.Conclusion The construction of eukaryotic expression vectors expressing shRNA sections targeting human MCHR2 and identification successfully established a favourable foundation for further study on the function of MCHR2.
