Suppression of the expression of exo- or endogenous genes by shRNA with the aid of PCR
- VernacularTitle:利用PCR方法获得shRNA抑制内外源性基因的表达
- Author:
Quan HONG
;
Di WU
;
Xiangmei CHEN
- Publication Type:Journal Article
- Keywords:
RNA, small interfering;
polymerase chain reaction;
fibronectins
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(09):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effects of shRNA produced by PCR on the suppression of the expression of exo- or endogenous genes. Methods The specific primers were designed, with which the shGFP-RNA and shFN-RNA were produced by PCR. The shGFP-RNA was transfected into 293T cell lines together with pEGFP plasmid, then the cells were detected by laser confocal microscopy 48h later. The shFN-RNA was transfected into rat mesenteric cell lines, then the cells were collected 48h later and the total RNA was extracted, which was reversely transcripted to cDNA. Then the expression level of FN mRNA was examined with real-time PCR, and the expression level of FN protein was examined with Western blot analysis. Results The results of laser confocal microscopy indicated that the EGFP could be successfully suppressed by shGFP-RNA produced by PCR; the results of real-time PCR and Western blot analysis revealed that FN expression level of the cells transfected with shFN-RNA was down regulated, and the level was much lower than those tansfected with independent shRNA (P
