Department of Applied Genomics, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan.
Kazusa Genome Technologies Inc., Kisarazu, Chiba, Japan.
PLoS One. 2024 Jul 5;19(7):e0302451. doi: 10.1371/journal.pone.0302451. eCollection 2024.
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.
即使使用先进的质粒和病毒载体，在不同转染细胞中达到多个基因的拷贝数也是具有挑战性的。我们使用转基因竞争系统（Kazusa cDNA 克隆和我们的双重组酶介导的盒交换系统的组合）在单个细胞中从单个拷贝基因实现了一个基因的表达。使用该系统，48 种核受体同时在一个培养皿中以相同的表达水平在 HEK293 中表达，并比较了细胞增殖率。在 8 周后，用 CMV 或 EF1 启动子驱动的 48 种核受体的表达转染的细胞之间观察到显著差异。EF1-NR1I2 细胞系在 2 至 8 周之间的增长最高，其增殖率比 EF1-DsRed 系高 1.13 倍。另一方面，在 8 周时表现出最大减少的 EF1-NR4A1 细胞系，其增殖率比 EF1-DsRed 系低 0.88 倍。这些结果在我们的转基因竞争系统和长期生长实验中都得到了证实。我们的转基因竞争系统提供了一种广泛、简单和准确的细胞竞争方法。
