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.
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http://dx.doi.org/10.1007/s11626-011-9463-y | DOI Listing |
Int J Mol Sci
December 2024
Medizinische Klinik und Poliklinik II und Lehrstuhl für Zelluläre Immuntherapie, Universitätsklinikum Würzburg, 97080 Würzburg, Germany.
The successful application of CAR-T cells in the treatment of hematologic malignancies has fundamentally changed cancer therapy. With increasing numbers of registered CAR-T cell clinical trials, efforts are being made to streamline and reduce the costs of CAR-T cell manufacturing while improving their safety. To date, all approved CAR-T cell products have relied on viral-based gene delivery and genomic integration methods.
View Article and Find Full Text PDFG3 (Bethesda)
October 2024
Laboratoire de Biologie et Modélisation de la Cellule, École Normale Supérieure de Lyon, CNRS UMR5239, Université Claude Bernard Lyon 1, 9 rue du Vercors, 69007 Lyon, France.
The cricket Gryllus bimaculatus is an emerging model insect of the order Orthoptera that is used in a wide variety of biological research themes. This hemimetabolous species appears highly complementary to Drosophila and other well-established holometabolous models. To improve transgenesis applications in G.
View Article and Find Full Text PDFZhejiang Da Xue Xue Bao Yi Xue Ban
October 2024
Department of Biophysics, School of Basic Medical Sciences, Zhejiang University, Hangzhou 310058, China.
Objectives: To establish a cell line stably expressing the transient receptor potential melastatin 2 (TRPM2) channel for screening TRPM2 inhibitors based on PiggyBac transposition system.
Methods: A plasmid PiggyBac-human TRPM2 (pPB-hTRPM2) eukaryotic expression vector was constructed using PiggyBac transposition system. The plasmid and a helper plasmid were co-transfected into HEK293T cells to express TRPM2, which was identified by fluorescence and patch-clamp assays.
Front Immunol
August 2024
Institute of Hematology and Blood Transfusion, Prague, Czechia.
Background: The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients.
View Article and Find Full Text PDFPLoS One
August 2024
GenomeFrontier Therapeutics TW Co., Ltd., Taipei City, Taiwan (R.O.C.).
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