Journal of Sericultural and Entomological Science (한국잠사곤충학회지)

Korean Society of Sericultural Science (한국잠사학회)
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Construction of Stably Transformed Bm5 Cells by Using Autographa californica Nuclear Polyhedrosis Virus IE1 Gene

  • Cho, Eun-Sook (College of Natural Resources and Life Science, Dong-A University, Pusan 604-714) ;
  • Jin, Byung-Rae (College of Natural Resources and Life Science, Dong-A University, Pusan 604-714) ;
  • Sohn, Hung-Dae (College of Natural Resources and Life Science, Dong-A University, Pusan 604-714) ;
  • Chol, Kwang-Ho (College of Natural Resources and Life Science, Dong-A University, Pusan 604-714) ;
  • Kim, Soung-Ryul (College of Natural Resources and Life Science, Dong-A University, Pusan 604-714) ;
  • Kang, Seok-Woo (Dept, of Sericulture & Entomology, The National Institute of Agricultural Science & Technology, RDA, Suwon 441-100 Korea.) ;
  • Yun, Eun-Young (Dept, of Sericulture & Entomology, The National Institute of Agricultural Science & Technology, RDA, Suwon 441-100 Korea.) ;
  • Kim, Sang-Hyun (Dept, of Sericulture & Entomology, The National Institute of Agricultural Science & Technology, RDA, Suwon 441-100 Korea.) ;
  • Kim, Keun-Young (Dept, of Sericulture & Entomology, The National Institute of Agricultural Science & Technology, RDA, Suwon 441-100 Korea.)
  • Published : 1998.12.01
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Abstract

To construct transformed 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% uncleotide 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/$m\ell$ G418 for two weeks. Individual clones were picked and each was amplified for further characterization. The genomic DNA from neomycin-resistnt cells was isolated and characterized by PCR using AcNPV IE1 gene-specific primers and by Southern blot analysis using neomycin gene probe. We concluded that AcNPV IE1 gene was functional in B. moridrived Bm5 cells as well as Spodaptera frugiperda-derived Sf9 cells to produce stably-transformed insect cells.

Keywords

References

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