A cell competition system with one gene expression from a single-copy gene in one cell

• Published in: PloS one Vol. 19; no. 7; p. e0302451
• Main Authors: Hasegawa, Yoshinori, Nakano, Megumi, Hosouchi, Tsutomu, Watanabe, Takashi, Yamaguchi, Izumi, Nakayama, Manabu, Ohara, Osamu
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 05.07.2024; Public Library of Science (PLoS)
• Subjects: Biology and Life Sciences; Cell culture; Cell growth; Cell proliferation; Cell Proliferation - genetics; Cloning; Competition; Expression vectors; Gene Dosage; Gene expression; Gene Expression - genetics; Genes; Genetic vectors; Genetic Vectors - genetics; Genomics; HEK293 Cells; Humans; Nuclear receptors; Plasmids; Promoter Regions, Genetic; Receptors; Receptors, Cytoplasmic and Nuclear - genetics; Receptors, Cytoplasmic and Nuclear - metabolism; Recombinase; Research and Analysis Methods; Scientific equipment and supplies industry; Transfection; Transgenes; United States
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0302451

Even with advanced plasmid and viral vectors, attaining copy numbers of multiple genes among different transfected cells is challenging. We achieved one gene expression from a single-copy gene in one cell using a transgene competition system, a combination of the Kazusa cDNA clones and our dual recombinase-mediated cassette exchange system. All 48 nuclear receptors were simultaneously expressed in one dish at the same expression level in HEK293 using this system, and the cell proliferation rate was compared. Significant differences were observed between cells transfected with CMV- or EF1 promoter-driven expression of the 48 nuclear receptors after 8 weeks. The EF1-NR1I2 cell line, which exhibited the highest increase from 2 to 8 weeks, showed 1.13-fold higher proliferation than the EF1-DsRed line. On the other hand, the EF1-NR4A1 cell line, which showed the maximum decrease at 8 weeks, showed 0.88-fold lower proliferation than the EF1-DsRed line. The results were confirmed in both our transgene competition system and long-term growth experiments. Our transgene competition system offers a wide-range, simple, and accurate cell competition method.