| Project/Area Number |
17510170
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Applied genomics
|
|---|
| Research Institution | FUJITA HEALTH UNIVERSITY |
|---|
Principal Investigator |
IKENO Masashi FUJITA HEALTH UNIVERSITY, Institute for Comprehensive Medical Science, Assistant Professor, 総合医科学研究所, 講師 (80298546)
|
|---|
| Project Period (FY) |
2005 – 2006
|
| Project Status |
Completed (Fiscal Year 2006)
|
| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,200,000 (Direct Cost: ¥2,200,000)
|
|---|
| Keywords | Gene transfer / Chromosome / Vector / Gene expression / Homologous recombination / HAC / Gene delivery / Transfection / 遺伝子 / 細胞・組織 / バイオテクノロジー |
|---|
| Research Abstract |
The main research objective is development of human artificial chromosome (HAC) vector for a new mammalian gene expression vector that promises carrying and delivering genes into recipient cells.
1. Development of technology for cloning large genes into HAC vector.
I developed technology for gene cloning into HAC vector by application of modifying BAC clones using Red/ET bacterial recombination and by application of inserting the modified BAC clones into HAC vector by Cre/lox recombination in mammalian cells. The genes carried on BACs could be systematically inserted into the HAC vector in many mammalian cells. The gene expression was analyzed using human STAT3 genomic gene cloned on HAC vector. STAT3 gene expressed at the same level of that of endogenous mouse STAT3 gene, and the expression level was retained after long culture of the cells containing HAC vectors. These results indicated that the HAC vector form chromosome structure on the inserted genes and mimic the cellular expression of the genes.
2. Development of technologies for purification and transfer of HAC vector
I established easy method for purification of HAC vector using recovery of mitotic chromosomes containing HAC vectors and using successive sucrose density gradient centrifugation. The HAC vector purified from CHO cells could be transferred into recipient human cells by DNA transfection method.
|
