title: Stable transformation and cloning mediated by piggyBac vector and RNA interference knockdown of Drosophila ovarian cell line.
abstract: An in vitro study is a powerful method for elucidating gene functions in cellular and developmental events. However, until date, no reliable in vitro transformation, cloning, or knockdown system has been reported for Drosophila cells, with the exception of S2 and Kc cells. In this study, we demonstrated that the piggyBac vector stably integrates donor DNA into ovarian somatic sheets derived from follicle stem cells. The transformed ovarian somatic sheet cells were easily cloned with a new piggyBac selection vector carrying enhanced green fluorescent protein and dihydrofolate reductase genes, egfp, and dhfr, respectively, in culture media containing methotrexate, an inhibitor of DNA synthesis. Donor egfp continued to be expressed at a high level in long-term culture. Furthermore, the translation of donor egfp was inhibited by treatment with double-stranded RNA derived from the target gene. The transfection and cloning methods mediated by the piggyBac vector would thus be useful for future analyses of gene functions in OSS cells and possibly be applicable to other Drosophila cell lines.
authors.affiliation: Department of Sciences, Faculty of Science, Ibaraki University, Mito, 310-8512, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Uetake
authors.forename: Kenji
authors.initials: K
authors.lastname: Oka
authors.forename: Yuzo
authors.initials: Y
authors.lastname: Niki
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004331:Drosophila melanogaster / Q000166:cytology* / Q000378:metabolism*; D005260:Female; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D010053:Ovary / Q000166:cytology*; D034622:RNA Interference; D014162:Transfection; D014170:Transformation, Genetic
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA
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