Construction and application of human artificial chromosome vectors for gene introduction using minichromosomes.
| Project/Area Number |
15590293
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Human genetics
|
|---|
| Research Institution | University of the Ryukyus |
|---|
Principal Investigator |
KANAME Tadashi University of the Ryukyus, Faculty of Medicine, Associate Prof., 医学部, 助教授 (40264288)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
NARITOMI Kenji University of the Ryukyus, Faculty of Medicine, Professor, 医学部, 教授 (20101446)
YANAGI Kumiko University of the Ryukyus, Faculty of Medicine, Instructor, 医学部, 助手 (90294701)
|
|---|
| Project Period (FY) |
2003 – 2004
|
| Project Status |
Completed (Fiscal Year 2004)
|
| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2004: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2003: ¥1,600,000 (Direct Cost: ¥1,600,000)
|
|---|
| Keywords | human artificial chromosomes / BAC / chromosome X / site specific recombination / gene therapy / DT40 / homologous recombination |
|---|
| Research Abstract |
We have established an efficient system for introducing a whole BAC clone including an intact gene unit onto a minichromosome based on the human X chromosome. The use of mutant loxP sites with Cre recombinase and direct selection for the retrofitting event led to a high efficiency of correctly retrofitted clones. A modified BAC containing the intact human HPRT gene was correctly retrofitted onto the minichromosome in DT40 cells in approximately 75% of analysed clones. Modification of BACs for retrofitting was used method previously reported (Kaname and Huxley, BioTechniques 31:273(2001)). For another BAC clone containing human factor IX gene, the efficiency of retrofitting was about 80%. For a plasmid (4.5kb) having a neoR expression unit, the retrofitting efficiency was 100%. The HPRT gene is expressed from the minichromosome at approximately the same level in both of these clones. This system allows one to introduce any gene or gene cluster which can be cloned as a BAC into the minichromosome for gene delivery.
We also tried to transfer the modified minichromosomes into other cells (HT1080,HPRT(-)) by microcell-fusion method. It was efficient to isolate the transchromosomic cells by screening of G418 resistant clones. Of the resistant clones, copy number of the minichromosome per cell was in range of 1-8. Its expression was, however, efficient and stable. Thus, the minichromosome would be used as a chromosome vector for gene therapy.
|
Report
(3 results)
Research Products
(13 results)
