| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
To analyze the function of genes, various methods for introducing genes to mammalian cells and tissues have been developed. Adenovirus vector is drawing attention, because it has high efficiency of introducing genes and it can be infected to non-dividing cells. However, as the viral genome is relatively long(36kb), in constructing vectors there are some problems of complicated procedures or remaining of replicapable viral particles. To overcom these problems, we developed a new constructing method by using cosmid for long DNA and Cre-loxP site-specific recombination system. In present study, we accomplished to develop more efficient and applicable method for constructing adenovirus vectors by adding improvements.
1. To insert longer expression cassettes, the cosmid containing adenoviral genome deleted E1 region(PALC) was deleted E3 region(PALCS). PALCS involving EGFP expression cassette was transfected to 293 cells, then replication of virus and the fluorescence of EGFP were observed. The efficient generation of adenovirus vectors and expression of introduced gene were recognized.
2. FLP-FRT, site-specific recombination system derived from yeast, like as Cre-loxP, was introduced to constructing adenovirus vectors. PAFCS generated using FRT sequences instead of loxP in pALC3 was investigated the efficiency of replicating adenovirus vectors. In same efficiency of pALC3, PAFCS could produce adenovirus vectors.
3. The expression cassette having DNA sequences interposed by loxP ligated to EGFP reporter gene was inserted to PAFCS. It was cotransfected to 293 cells with FLP and Cre recombinase expressing plasmids, and virus replication and EGFP fluorescence were observed. The results showed that this method could control the expression of introduced gene as required. It will be useful to introduce genes of cytotoxic protein, silently at first.
More efficient and applicable method for constructing adenovirus vectors was developed by improvements accomplished in this study.
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