Construction of Thymosin ?4 Shrna Lentivirus and Its Effects on the Expression of Thymosin ?4 in 293T Cells
- VernacularTitle:人胸腺素?_4 RNA干扰载体的构建及其对293T细胞目的基因表达的影响
- Author:
Sijian HUANG
;
Chang LIU
;
Zelian QIN
- Publication Type:Journal Article
- Keywords:
Thymosin ?4;
293T cell;
RNA interference;
Lentivirus
- From:
Chinese Journal of Minimally Invasive Surgery
2005;0(07):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a recombinant lentiviral vector for human thymosin ?4 (TMSB4) and to test the mRNA and protein expression of TMSB4 in 293T cells after being infected by shRNA lentivirus. Methods We designed a specific sequence of small hair RNA targeting TMSB4 gene; the complementary DNA containing both sense and antisense oligo DNA of the targeting sequence was cloned into the pGCSIL-GFP vector to construct a lentiviral vector. The vector was converted into the competent DH5a coli,which confirmed by PCR and sequencing. Then the viral vector and the packed systemic vector were cotransfected 293T cells to get the lentivirus. The virus titer was determined. Afterwards,in the 293T cells infected with the lentivirus,the expression of TMSB4 was detected by real time PCR and immunocytochemistry. Similarly,a primary fibroblast was also infected with the lentivirus. Results Compared with negative control cells,the mRNA and protein levels of TMSB4 in 293T cells infected with the lentivirus were reduced by 44% (0.56?0.11 vs 1.00?0.06,F=89.673,P
