Department of Cell and Developmental Biology, University of Illinois, Urbana, IL, USA.
Biology Department, College of the Ozarks, Point Lookout, MO, USA.
Gene Ther. 2018 Aug;25(5):376-391. doi: 10.1038/s41434-018-0021-z. Epub 2018 Jun 21.
Reproducible and stable transgene expression is an important goal in both basic research and biotechnology, with each application demanding a range of transgene expression. Problems in achieving stable transgene expression include multi-copy transgene silencing, chromosome-position effects, and loss of expression during long-term culture, induced cell quiescence, and/or cell differentiation. Previously, we described the "BAC TG-EMBED" method for copy-number dependent, chromosome position-independent expression of embedded transgenes within a BAC containing ~170 kb of the mouse Dhfr locus. Here we demonstrate wider applicability of the method by identifying a BAC and promoter combination that drives reproducible, copy-number dependent, position-independent transgene expression even after induced quiescence and/or cell differentiation into multiple cell types. Using a GAPDH BAC containing ~200 kb of the human GAPDH gene locus and a 1.2 kb human UBC promoter, we achieved stable GFP-ZeoR reporter expression in mouse NIH 3T3 cells after low-serum-induced cell cycle arrest or differentiation into adipocytes. More notably, GFP-ZeoR expression remained stable and copy-number dependent even after differentiation of mouse ESCs into several distinct lineages. These results highlight the potential use of BAC TG-EMBED as an expression platform for high-level but stable, long-term expression of transgene independent of cell proliferative or differentiated state.
可重现且稳定的转基因表达是基础研究和生物技术的重要目标，每个应用都需要一系列的转基因表达。实现稳定的转基因表达的问题包括多拷贝转基因沉默、染色体位置效应以及在长期培养、诱导细胞静止和/或细胞分化过程中表达的丧失。此前，我们描述了“BAC TG-EMBED”方法，用于在包含约 170kb 小鼠 Dhfr 基因座的 BAC 中嵌入的转基因的拷贝数依赖性、染色体位置独立性表达。在这里，我们通过鉴定一个 BAC 和启动子组合，证明了该方法的更广泛适用性，即使在诱导静止和/或细胞分化为多种细胞类型后，该组合仍能驱动可重现的、拷贝数依赖性、位置独立性的转基因表达。使用包含约 200kb 人 GAPDH 基因座的 GAPDH BAC 和 1.2kb 人 UBC 启动子，我们在低血清诱导的细胞周期阻滞或分化为脂肪细胞的小鼠 NIH 3T3 细胞中实现了 GFP-ZeoR 报告基因的稳定表达。更值得注意的是，即使在将小鼠 ESCs 分化为几种不同的谱系后，GFP-ZeoR 表达仍然稳定且拷贝数依赖性。这些结果突出了 BAC TG-EMBED 作为表达平台的潜在用途，可用于高水平但稳定、长期的转基因表达，而与细胞增殖或分化状态无关。
