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KMID : 1041219980400010052
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Journal of Sericultural and Entomological Science
1998 Volume.40 No. 1 p.52 ~ p.59
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Construction of Recombinant Bombyx mori Nuclear Polyhedrosis Virus Using a FLP/FRT System of Yeast, Saccharomyces cerevisiae 2 ㎛ plasmid
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강석우/Kang, Seok Woo
윤은영/김상현/김근영/한명세/강석권/Yun, Eun Young/Kim, Sang Hyun/Kim, Keun Young/Han, Myung Sae/Kang, Seok Kwon
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Abstract
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For the construrtion of plasmid and BmNPV carrying the FRT recognition site for the FLP recombinases, we synthesized the wild type FRT oligonucleotides. The target FRT sequences consist of three 13bp repeated DNA sequences; two repeats in a direct orientation and one inverted relative to the other two. In addition, there is an 8bp spacer region between the repeats which determine the orientation of the FRT recombination site. In order to place the FRT site both in the target BmNPV genome and the transfer vector, we constructed a plasmid, pFRTβ-gal, carrying the FRT sites within the cloning sites of pSV vector and a recombinant BmNPV, vFRTPH, carrying the FRT sites at a downstream of polyhedrin promoter, respectively. In order to test the functionality of the FLP/FRT site-specific recombination system, vFRTPH, pFRTβ-gal and pHsFLP DNA were co-transfected into BmN-4 cells. The resulting recombinant virus was designated a vFRT2β-gal. From construction analysis of the vFRT2 β-gal with PCR technique it was concluded that the entire pFRTβ-gal Plasmid with β-galactosidase gene and origines of replication flanked by two functional hybrid FRT sequences. The efficiency of recombination was 8.7%, which was higher than that(2.2 %) of recombination between a conventional transfer vector and the wild type BmNPV.
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