Department of Pharmacology, Robert Wood Johnson Medical School, Cancer Institute of New Jersey, USA
Mol Imaging. 2009 Dec;8(6):305-18.
Bicistronic vectors are useful tools for exogenous expression of two gene products from a single promoter element; however, reduced expression of protein from the second cistron compared with the first cistron is a common limitation to this approach. To overcome this limitation, we explored use of dihydrofolate reductase (DHFR) complementary DNA encoded in bicistronic vectors to induce a second protein of interest by methotrexate (MTX) treatment. Previous studies have demonstrated that levels of DHFR protein and DHFR fusion protein can be induced translationally following MTX treatment of cells. We demonstrated that in response to MTX treatment, DHFR partner protein in a bicistronic construct is induced for longer periods of time when compared with endogenous DHFR and DHFR fusion protein, in vitro and in vivo. Using rapamycin pretreatment followed by MTX treatment, we also devised a strategy to modulate levels of two proteins expressed from a bicistronic construct in a cap-independent manner. To our knowledge, this is the first report demonstrating that levels of proteins in DHFR-based bicistronic constructs can be induced and modulated using MTX and rapamycin treatment.
双顺反子载体是从单个启动子元件中外源表达两种基因产物的有用工具；然而，与第一个顺式元件相比，第二个顺式元件的蛋白表达减少是该方法的一个常见限制。为了克服这一限制，我们探索了使用双顺反子载体中编码的二氢叶酸还原酶 (DHFR) cDNA 通过甲氨蝶呤 (MTX) 处理诱导第二个感兴趣的蛋白质。先前的研究表明，在 MTX 处理细胞后，DHFR 蛋白和 DHFR 融合蛋白的水平可以通过翻译进行诱导。我们证明，与内源性 DHFR 和 DHFR 融合蛋白相比，在体外和体内，双顺反子构建体中的 DHFR 伴侣蛋白在 MTX 处理后可以诱导更长时间。通过预先用雷帕霉素处理，然后用 MTX 处理，我们还设计了一种策略，以非帽依赖性方式调节来自双顺反子构建体表达的两种蛋白质的水平。据我们所知，这是第一个证明可以使用 MTX 和雷帕霉素处理诱导和调节基于 DHFR 的双顺反子构建体中蛋白质水平的报告。
