1994 Fiscal Year Final Research Report Summary
ANALYSIS OF ANTI-ATHEROGENIC MECHANISM DUE TO CELLS TRANSFECTED
| Project/Area Number |
05670871
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
内分泌・代謝学
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
KOBORI Sozo SCHOOL OF MEDICINE,ASSISTANT PROFESSOR, 医学部・附属病院, 講師 (00201492)
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| Co-Investigator(Kenkyū-buntansha) |
MIYATA Takao SCHOOL OF MEDICINE,RESEARCH ASSOCIATE, 医学部, 助手 (70244118)
KISHIKAWA Hideki SCHOOL OF MEDICINE,RESEARCH ASSOCIATE, 医学部・附属病院, 助手 (30161441)
SHICHIRI Motiaki SCHOOL OF MEDICINE,PROFESSOR, 医学部, 教授 (00028515)
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| Project Period (FY) |
1993 – 1994
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| Keywords | Human LDL receptor gene / Transfection / Electronporation / PVS2-neo / CHO cells / HepG2 cells |
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| Research Abstract |
Transfection of human low density lipoprotein receptor gene (pLDLR-2) to CHO cells and HepG2 cells
1.Transfection
Method of the Electronporation was performed to CHO cells or HepG2 cells using plasmid with human LDL receptor cDNA (pLDLR-2) and PVS2-neo under the condition with voltage 0.2kV and time constant 18 msec.
2.Selection of transfected cells
1) In CHO cells, neomycin-resistant colonies, stable colonies were obtained accroding to the method.
2) In HepG2 cells, transient colonies could be obtained, but stable colonies could not.
3.Identification of transfected cells
1) Binding assay
In the experiments of binding assay with 1251-labeled LDL,affimity and binding capacity were markedly increased in both CHO cells and HepG2 cells transfected as compared with wild type CHO cells and HepG2 cella.
2)Western blotting
Membrane pellets from CHO-pLDL-R2 or HepG2-pLDL-R2 were subjected to SDS-polyacrylamide gel electrophoresis in 3-12% gel and then electrophoretically transferred to nitrocellulose paper. Immunoblotting was performed using 10 mg.ml of anti-human LDL receptor monoclonal antibody, followed by 1251-labeled protein A.Ligand blotting detected a single protein (around 13 kDa) in membrane pellets of these cells.
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