Construction of si-RNA synthesis plasmids targeting human Hsp90? gene and observation of their transfection efficiencies
- VernacularTitle:人热休克蛋白90?基因沉默质粒的构建及转染效率的观察
- Author:
Yewei JI
- Publication Type:Journal Article
- Keywords:
Hsp90;
RNAi;
Gene transfection;
Stress
- From:
Journal of Chongqing Medical University
1987;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct the plasmid that can direct the synthesis of si-RNA like transcripts to specifically and effectively in hibit the mRNA level of human Hsp90?gene,and to compare the transfection efficiencies of the plasmids in different cell strains. Methods:Three 64 nt oligos corresponding to different regions in the target gene are chemically synthesized and annealed,then inserted into pSUPER EGFP1,proliferated by DH5a,and determined by endodigestion and sequencing. Three strains,HepG2、HUVEC and HeK293,were cultured. We then transfected the plasmids into the cells under different ratio of plasmid and Lipofectamine,and compared the transfection efficiencies of them by detection of EGFP Fluoresence. Results:The presence of positive recombinant clones was verified by double-digestion and sequencing. The bases inserted into the pasmids were correct.And the positive colonies were named as pSuper-Hsp90?1、pSuper-Hsp90?2 and pSuper-Hsp90?3. and the same condition,the constructed plasmid had the highest transfection efficiency in HeK293,and the lowest in HepG2 cells. Conclusions:The si-RNA synthesis plasmids targeting Hsp90? could be successfully constructed by using psuper plasmids,and the difference of their transfection efficiencies in different cells was significant.
