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KMID : 1041219980400020111
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Journal of Sericultural and Entomological Science
1998 Volume.40 No. 2 p.111 ~ p.116
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Construction of Stably Transformed Bm 5 Cells by Using Autographa californica Nuclear Polyhedrosis Virus IE 0 Gene
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Cho, Eun Sook/Cho, Eun Sook
Jin, Byung Rae/Sohn, Hung Dae/Choi, Kwang Ho/Kim, Soung Ryul/Kang, Seok Woo/Yun, Eun Young/Jin, Byung Rae/Sohn, Hung Dae/Choi, Kwang Ho/Kim, Soung Ryul/Kang, Seok Woo/Yun, Eun Young
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Abstract
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To construct transfurmed Bm5 cells, Autographa californica nuclear polyhedrosis virus(AcNPV) IE1 gene, an immediate early viral gene was firstly used in this study. AcNPV IE1 gene, which shares on 95.3% nucleotide sequence homology with Bombyx mori nuclear polyhedrosis virus (BmNPV) IE1 gene, was isolated and cloned into pBluescript. Neomycin gene from pKO-neo was inserted under the control of the IE1 promoter to yield pAcIE1-neo. The plasmid pAcIE1-neo was transfected into Bm5 or Sf9 cells, and neomycin-resistant cells were selected in TC100 medium containing 10% fetal bovine serum (FBS) and 1 mg/ml G4l8 for two weeks. Individual clones were picked and each was amplified for further characterization. The genomic DNA from neomycin-resistant cells was isolated and characterized by PCR using AcNPV IEI gene-specific primers and by Southern blot analysis using neomycin gene probe. We concluded that AcNPV IE1 gene was functional in B. mori-derived Bm5 cells as well as Spodoptera fugjprrda-derived Sf9 cells to produce stably-transformed insect cells
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