Construction and gene silencing evaluation of S6K1 shRNA gene recombinant adenovirus
10.3760/cma.j.issn.0254-5101.2010.05.004
- VernacularTitle:S6K1 shRNA基因重组腺病毒的构建及对基因沉默效果的研究
- Author:
Shuying LI
;
Demin YU
;
Wenyan MIU
;
Zhi YAO
- Publication Type:Journal Article
- Keywords:
S6K1;
shRNA;
Viral vector;
Gene silencing
- From:
Chinese Journal of Microbiology and Immunology
2010;30(5):405-409
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct S6K1 shRNA gene recombinant adenovirus(S6K1 Ax)and evaluate its gene silencing effects on mouse cell lines and C57 BL/6J mice level.Methods Three S6K1 shRNA gene sequences were designed and spliced from pcPUR plasmid,pcDNA3.1 plasmid to cosmid plasmid and transfected into adenovirus.S6K1 Ax which has best gene silencing effect was selected and proliferated in 293 cell.Silencing effect of S6K1Ax was checked on mice AML12,C2C12,3T3-L1 cell lines.C57BL/6J liver was obtained after S6K1 Ax was injected into mice tail vein six days later.S6K1 was evaluated by qRT-PCR and Western blot.Alanine transferanse(ALT)was examined before and after S6K1 Ax injected.Results S6K1Ax can silence S6K1 expression of mouse AML12,C2C12 and 3T3-L1 cell lines and liver of C57 BL/6J mice on Western blot.S6K1 mRNA expression of C57BL/6J liver were control group 1.39±0.21 vs S6K1Ax group 0.63±0.09,t=6.132.P<0.01.ALT of mice hepatic function did not change after S6K1Ax injected:before(15.15±4.43)U/L,after(17.32±4.22)U/L,t=1.451,P>0.05.Conclusion Construction of shS6K1 Ax can knockdown S6K1 gene on mice cell lines and C57BL/6J mice liver,it provides a good tool to study the function of S6K1.
