| Project/Area Number |
12672145
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
|
|---|
| Research Institution | National Institute of Health Sciences |
|---|
Principal Investigator |
HAYAKAWA Takao National Institute of Health Sciences, Division of Biological Chemistry and Biologicals, Division Director, 生物薬品部, 部長 (50124392)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
MIZUGUCHI Hiroyuki National Institute of Health Sciences, Division of Biological Chemistry and Biologicals, Researcher, 生物薬品部, 研究員 (50311387)
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|---|
| Project Period (FY) |
2000 – 2001
|
| Project Status |
Completed (Fiscal Year 2001)
|
| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | gene therapy / adenovirus vector / adeno-associated virus vector / gene requlation / plasmid vector |
|---|
| Research Abstract |
In basic genetic research, control oftransgene expression in mammalian cells should be desirable forgene therapyand the studyofgene function. Permanentand higher levels of gene expression should be also preferable. The expression levels of the introduced gene depend mostly on the strength oftranscriptional regulatory elements and the transduction efficiency of the gene transfer vector.
In this study, we investigated the effective combination of commonly-used regulatory elements, such as the promoter/enhancer, intron, and polyadenylation signal (P(A)) sequence by constructing a series of plasmids and recombinant adenovirus vectors that differed only in the particular sequence element being evaluated. Of the several promoter/enhancers that were tested, hybrid CA promoter/enhancer containing human cytomegalovirus immediate-early 1 gene (CMV) enhancer and chicken β-actin promoterwith the β-actin intron sequence, and the improved CMV promoter/enhancer containing the largest intron of CMV(int
… More
ron A) produced the highest levels of expression both in vitro and in vivo. P(A)sequences were found to have significant effects on transgene expression. These comparative analyses could provide a systematic reference for the development of vector construction for gene therapy, vaccine development, and gene transfer experiments.
Next, we examined the stable gene expression using the plasmids containing the components derived from adeno-associated virus (AAV). We examined the efficiency of colony formation of the neomycin resistant cells which were transfected with the plasmid containing neomycin resistant gene franked with AAVITR (inverted terminal repeat). Number of colony of neomycin resistant cells transfected with the plasmid with AAVITR and Rep78(AAVderived proteinj-expressing plasmid was much higher than that with the plasmid without AAVITRand Rep78expressing plasmid, suggesting that Rep78 protein assisted the integration of the foreing gene intothe host genome. We are now applying this techiniqes intothe integration of the genomic foreign DNA into the host genome. This system using genomic DNA could get stable and regulated gene expression. Less
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