Department of Biophysics and Biochemistry, The University of Tokyo, Tokyo 113-0033, Japan.
G3 (Bethesda). 2012 Jul;2(7):741-51. doi: 10.1534/g3.112.002832. Epub 2012 Jul 1.
Although various genetic tools have been developed and used as transgenes, the expression of the transgenes often is hampered by negative regulators. Disrupting such negative regulators of gene expression is potentially a way to overcome the common problem of low expression of transgenes. To find such regulators whose mutations enhance transgene expression in Caenorhabditis elegans, we took advantage of a newly developed reporter transgene, lin-11pAΔ::venus. This transgene induces expression of a fluorescent protein, Venus, in specific neurons including AIZ, where the expression was stochastic. The frequency of reporter expression in AIZ seemed to be correlated with the strength of transgene expression. By using this system, in which a moderate increase of expression was converted to all-or-none expression states, we describe here a forward genetic screen for mutations that enhance the expression of transgenes. Through the screen, we found that mutations in the pqe-1 gene, which encodes a Q/P-rich nuclear protein with an exonuclease domain, increase the chance of reporter expression in AIZ. The fluorescence intensity in RIC, in which all lin-11pAΔ::venus animals show reporter expression, was increased in pqe-1 mutants, suggesting that pqe-1 reduces the expression level of the transgene. Expression of transgenes with other promoters, 3'UTR, or reporter genes was also enhanced by the pqe-1 mutation, suggesting that the effect was not specific to a particular type of transgenes, whereas the effect did not seem to extend to endogenous genes. We propose that pqe-1 mutants can be used to increase the expression of various useful transgenes.
虽然已经开发并使用了各种遗传工具作为转基因，但转基因的表达通常受到负调控因子的阻碍。破坏这种基因表达的负调控因子可能是克服转基因表达普遍较低的一种方法。为了找到能够增强秀丽隐杆线虫中转基因表达的这种负调控因子，我们利用了一种新开发的报告基因转基因 lin-11pAΔ::venus。该转基因在包括 AIZ 在内的特定神经元中诱导荧光蛋白 Venus 的表达，其表达是随机的。报告基因在 AIZ 中的表达频率似乎与转基因表达的强度相关。利用这个系统，我们将中等程度的表达增加转化为全有或全无的表达状态，在这里我们描述了一个用于筛选增强转基因表达的突变的正向遗传学筛选。通过筛选，我们发现 pqe-1 基因突变，该基因编码具有外切酶结构域的 Q/P 丰富核蛋白，增加了报告基因在 AIZ 中表达的机会。在 RIC 中，所有 lin-11pAΔ::venus 动物都显示报告基因表达，pqe-1 突变体中的荧光强度增加，表明 pqe-1 降低了转基因的表达水平。其他启动子、3'UTR 或报告基因的转基因表达也被 pqe-1 突变增强，这表明这种效应不是特定于特定类型的转基因，而这种效应似乎不会扩展到内源性基因。我们提出 pqe-1 突变体可用于增强各种有用的转基因的表达。
