| Project/Area Number |
10470505
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Human genetics
|
|---|
| Research Institution | Osaka University |
|---|
Principal Investigator |
KANEDA Yasufumi Graduate School of Medicine, Osaka University, Professor, 医学系研究科, 教授 (10177537)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
SAEKI Yoshinaga Graduate School of Medicine, Osaka University, Assistant Professor, 医学系研究科, 助手 (20314320)
田中 亀代次 大阪大学, 細胞生体工学センター, 教授 (80144450)
|
|---|
| Project Period (FY) |
1998 – 1999
|
| Project Status |
Completed (Fiscal Year 1999)
|
| Budget Amount *help |
¥12,900,000 (Direct Cost: ¥12,900,000)
Fiscal Year 1999: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 1998: ¥8,000,000 (Direct Cost: ¥8,000,000)
|
|---|
| Keywords | Gene insertion / Gene delivery / HVJ-liposomes / gene expression / transposon / transposase / 遺伝子挿入 / アデノ随伴ウイルス / 遺伝子維持 / 長期遺伝子発現 |
|---|
| Research Abstract |
The objective of this study is the development of gene therapeutics for genetic disorders caused by one-point mutation. Mutations of xeroderma pigmentosum group (A) (XPA) have been well chracterized. Using XPA as a model, we attempted to insert genes to specific sites of chromosomes. According to the correction of a point mutation using DNA/RNA chimeric oligonucleotides described by Kmiec, E., we constructed 68 mer chimeric DNA/RNA oligonucleotides containing normal sequence of the boundary of the intron 3 and exon 4 of the human XPA gene because the conversion from G to C occurs in XP2OSSV (XPA) cells. The chimeric oligonucleotides were transferred to XP2OSSV (XPA) cells using HVJ-liposomes which have been evalutated as the most efficient vehicle for introducing oligonucleotides into cells. After transfer, cells were subjected to UV-irradiation. However, no colonies appeared. Then, we tried to detect the correction of the point mutation in XP2OSSV (XPA) cells by PCR without UV-irradiation. None of 200000 cells showed the correction of the mutation. We concluded that the chimeric oligonucleotides were not so effective for the correction of point mutation as in the previous reports. Next, we attempted to develop the methods for increasing the insertion of a transgene into host chromosomes. One of the strategies was the use of transposon/transposase system derived from fish. Using this system, neo-resistant stable transformants were obtained approximately 30 fold more efficiently in cultured HeLa cells than that without the transposase. Then, we transferred neo-resistant gene with the transposon/transposase to mouse liver using HVJ-liposomes. Neo-resistant gene was detected in the liver by PCR for more than 6 weeks whereas it disappeared in 2 weeks without the transposase. This system should be more extensively evaluated for gene insertion into tissue cells, but it seems to be promising for long-term gene expression and the replacement of mutant genes.
|
