title: Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture.
abstract: A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus that are involved in integration of the densovirus in insect cell chromosomes and a promoter/enhancer system that results in high levels of expression. The plasmid also contains two markers that permit selection of transformed insect cells by antibiotic resistance or by cell-sorting for fluorescent protein expression. Transformation of Bombyx mori Bm5 or Spodoptera frugiperda Sf9 cultured cells with the pDP9 vectors results in the integration of the pDP9 plasmid into genomic DNA of Bm5 and Sf9 cells. pDP9 contains a multiple cloning site (MCS) 3' of the densoviral P9 promoter and insertion of a protein coding sequence within the MCS results in high level expression by pDP9 transformed cells. P9 driven transcription in the pDP9 transformed Sf9 cells produced foreign gene transcript levels that were 30 fold higher than actin 3 driven transgenes and equivalent to hr5IE1 driven transgenes. The pDP9 vector transformation results in the efficient selection of clones for assessment of promoter activity.
authors.affiliation: Center for Medical, Agricultural and Veterinary Entomology, Agricultural Research Service, US Department of Agriculture, 1700 SW 23rd Drive, Gainesville, FL 32608, USA. Electronic address: paul.shirk@ars.usda.gov.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Shirk
authors.affiliation: Center for Medical, Agricultural and Veterinary Entomology, Agricultural Research Service, US Department of Agriculture, 1700 SW 23rd Drive, Gainesville, FL 32608, USA.
authors.forename: Richard B
authors.initials: RB
authors.lastname: Furlong
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D012831:Bombyx / Q000166:cytology / Q000235:genetics*; D002478:Cells, Cultured; D059006:Chromosomes, Insect; D017990:Densovirus / Q000235:genetics; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics; D004625:Embryo, Nonmammalian; D018628:Gene Dosage; D005786:Gene Expression Regulation; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D018411:Spodoptera / Q000166:cytology / Q000235:genetics*; D014162:Transfection; D019076:Transgenes
chemical_list: D005819:Genetic Markers; D049452:Green Fluorescent Proteins
keywords: Bombyx mori Bm5 cells; Densovirus integration; Expression; Genetic transformation; Spodoptera frugiperda Sf9 cells; Transfection