Chapman A B, Costello M A, Lee F, Ringold G M
Mol Cell Biol. 1983 Aug;3(8):1421-9. doi: 10.1128/mcb.3.8.1421-1429.1983.
Mouse 3T6 cells were transformed with a chimeric DNA plasmid, pSVMgpt, in which the mouse mammary tumor virus (MMTV) promoter was fused to the Escherichia coli gene encoding xanthine-guanine phosphoribosyl transferase (Eco gpt). The transformants exhibited glucocorticoid-inducible expression of Eco gpt. With limiting xanthine concentrations, conditions were established in which cell growth became hormone dependent. Cells selected for their ability to grow in limiting concentrations of both xanthine and glucocorticoids contained amplified levels of Eco gpt DNA, and expression of Eco gpt remained glucocorticoid inducible in these amplified cells. Thus, amplification of the MMTV promoter region in itself did not abolish hormonal responsiveness of a gene. In addition to increased levels of Eco gpt DNA, some of the selected cells also exhibited increased levels (two- to threefold) of glucocorticoid receptors. Lastly, we found that excessive expression of Eco gpt is toxic to 3T6 cells; by maintaining low hormone levels and, therefore, low levels of expression, we were able to select cells with amplified Eco gpt. Thus, the MMTV promoter may be of general utility in expressing genes whose products may be lethal if they are produced in excessive quantities.
用嵌合DNA质粒pSVMgpt转化小鼠3T6细胞，该质粒中鼠乳腺肿瘤病毒（MMTV）启动子与编码黄嘌呤 - 鸟嘌呤磷酸核糖基转移酶的大肠杆菌基因（Eco gpt）融合。转化体表现出Eco gpt的糖皮质激素诱导型表达。在黄嘌呤浓度受限的情况下，建立了细胞生长变为激素依赖性的条件。选择在黄嘌呤和糖皮质激素浓度均受限的情况下仍能生长的细胞，其Eco gpt DNA水平扩增，并且在这些扩增细胞中Eco gpt的表达仍受糖皮质激素诱导。因此，MMTV启动子区域的扩增本身并未消除基因的激素反应性。除了Eco gpt DNA水平升高外，一些选择的细胞还表现出糖皮质激素受体水平升高（两到三倍）。最后，我们发现Eco gpt的过度表达对3T6细胞有毒性；通过维持低激素水平，从而维持低表达水平，我们能够选择Eco gpt扩增的细胞。因此，MMTV启动子在表达其产物如果过量产生可能具有致死性的基因方面可能具有普遍用途。
