Construction of recombinant adenovirus carrying GRIM19 and its effect on SW480 cells.
In order to examine the effect of GRIM19 on colon cancer cell SW480, the recombinant adenovirus carrying GRIM19 gene was constructed and transfected into SW480 cells. GRIM19 cDNA was amplified by PCR with the template pcxn2-GRIM19 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 digestion. After transfection of linearized pAd-GRIM19 with PacI into HEK293 cells, Ad-GRIM19 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 increased 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.
Wang
Shen
S
Cancer Research Institute/Department of General Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. wangshenll2000@yahoo.com.cn
Wang
Guihua
G
Deng
Yu
Y
Luo
Xuelai
X
Li
Xiaolan
X
Tao
Deding
D
Gong
Jianping
J
Hu
Junbo
J
D000256:Adenoviridae / Q000378:metabolism*; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics / Q000628:therapy*; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry; D005821:Genetic Techniques; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D008954:Models, Biological; D008957:Models, Genetic; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D013997:Time Factors
D051017:Apoptosis Regulatory Proteins; D009247:NADH, NADPH Oxidoreductases; C439057:NDUFA13 protein, human