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 pAdTrac...

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Bibliographic Details
Published in:Journal of Huazhong University of Science and Technology. Medical sciences 2008-02, Vol.28 (1), p.14-16
Main Author: 王坤 王桂华 邓豫 罗学来 李小兰 陶德定 龚建平 胡俊波
Format: Article
Language:English
Subjects:
Adenoviridae - metabolism
Adenovirus
Apoptosis
Apoptosis Regulatory Proteins - biosynthesis
Apoptosis Regulatory Proteins - physiology
Cell Line
Cell Line, Tumor
Colonic Neoplasms - genetics
Colonic Neoplasms - therapy
Electrophoresis, Agar Gel
Flow Cytometry
Genetic Techniques
Genetic Therapy - methods
GRIM-19
Humans
Medicine
Medicine & Public Health
Models, Biological
Models, Genetic
NADH, NADPH Oxidoreductases - biosynthesis
NADH, NADPH Oxidoreductases - physiology
Plasmids - metabolism
SW480细胞
Time Factors
基因表达
腺病毒
重组体
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Summary: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.
ISSN:1672-0733
1993-1352
DOI:10.1007/s11596-008-0104-y