Construction of Recombinant Adenovirus Carrying GRIM19 and Its Effect on SW480 Cells
- Author:
WANG SHEN
1
;
WANG GUIHUA
;
DENG YU
;
LUO XUELAI
;
LI XIAOLAN
;
TAO DEDING
;
GONG JIANPING
;
HU JUNBO
Author Information
1. 华中科技大学同济医学院附属同济医院
- Keywords:
GRIM-19 gene;
recombinant adenovirus;
SW480 cells;
cell death
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2008;28(1):14-16
- CountryChina
- Language:Chinese
-
Abstract:
In order to examine the effect of GRIM 19 on colon cancer cell SW480, the recombinant adenovirus carrying GRIM19 gene was constructed and transfected into SW480 cells. GRIMI9 cDNA was amplified by PCR with the template pcxn2-GRlMl9 and cloned into the shuttle plasmid pAdTrack-CMV. The plasmid pAdTrack-CMV-GRIM19 was linearized by PmeI and homologously recombined with bone plasmid pAdEasy-1 in BJ5183, followed by identification by enzyme diges- tion. After transfection of linearized pAd-GRIM19 with PacI into HEK293 cells, Ad-GRIMI9 was obtained and amplified by 3 circles. SW480 cells were infected with Ad-GRIM19. The apoptosis rate was detected by flow cytometry. Agarose electrophoresis revealed the bands of recombinant plasmids identified by enzyme digestion were in the right range corresponding with expectation. Under the fluorescent microscopy, the package of Ad-GRIM19 in HEK293 cells and the expression of Ad-GRIM19 in SW480 cells were observed. The transfection of Ad-GRIM19 into SW480 cells in-creased the apoptosis rate of SW480 cells as compared with controls. It was concluded that Ad-GRIM19 was successfully constructed and the overexpression of GRIM19 in colon cancer cell lines could promote the apoptotic cell death.
