Stable and reproducible transgene expression independent of proliferative or differentiated state using BAC TG-EMBED.
Stable and reproducible transgene expression independent of proliferative or differentiated state using BAC TG-EMBED.
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DOI：
10.1038/s41434-018-0021-z
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发表时间：
2018-08
期刊：
影响因子：
5.1
通讯作者：
Belmont AS
中科院分区：
文献类型：
--
作者：
Chaturvedi P;Zhao B;Zimmerman DL;Belmont AS
可重复和稳定的转基因表达是基础研究和生物技术的重要目标，每种应用都需要一系列转基因表达。实现稳定转基因表达的问题包括多拷贝转基因沉默、染色体位置效应和长期培养期间的表达丧失、诱导的细胞静止和/或细胞分化。先前，我们描述了“BAC TG-EMBED”方法，用于在含有约170 kb小鼠Dhfr基因座的BAC内嵌入转基因的拷贝数依赖性、染色体位置无关性表达。在这里，我们通过鉴定BAC和启动子组合来证明该方法的更广泛的适用性，该BAC和启动子组合即使在诱导静止和/或细胞分化成多种细胞类型后也能驱动可再现的、拷贝数依赖性的、位置非依赖性的转基因表达。使用含有~200 kb人GAPDH基因座和1.2 kb人UBC启动子的GAPDH BAC，我们在低血清诱导的细胞周期停滞或分化为脂肪细胞后，在小鼠NIH 3 T3细胞中实现了稳定的GFP-ZeoR报告基因表达。更值得注意的是，GFP-ZeoR的表达保持稳定和拷贝数依赖性，即使在小鼠胚胎干细胞分化成几个不同的谱系。这些结果突出了BAC TG-EMBED作为表达平台的潜在用途，该表达平台用于高水平但稳定的、长期的转基因表达，而不依赖于细胞增殖或分化状态。
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.
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DOI：
10.1073/pnas.93.17.9067
发表时间：
1996-08-20
影响因子：
11.1
作者：
Bronson, SK;Plaehn, EG;Smithies, O
通讯作者：
Smithies, O
影响因子：
12.4
作者：
Chen, Zhi-Ying;Riu, Efren;Kay, Mark A.
通讯作者：
Kay, Mark A.
影响因子：
2.7
作者：
de Boer, L;Gray, PP;Sunstrom, NA
通讯作者：
Sunstrom, NA
影响因子：
5.3
作者：
Cherry, SR;Biniszkiewicz, D;Jaenisch, R
通讯作者：
Jaenisch, R
影响因子：
20.3
作者：
Burns, William R.;Zheng, Zhili;Morgan, Richard A.
通讯作者：
Morgan, Richard A.