Novel expression cloning methods based on retrovirus mediated gene transfer.

我们之前建立了一种高效的逆转录病毒介导的表达克隆方法。利用这个系统，我们现在已经开发了一种新的表达克隆方法，在这种方法中，cdna可以根据其产物的亚细胞定位来分离。在pMX逆转录病毒载体中，利用随机六聚体合成cDNA，并将其融合到绿色荧光蛋白（GFP） cDNA的5'端，构建cDNA-GFP融合逆转录病毒文库。然后将衍生的逆转录病毒感染到NIH3T3细胞中，根据其蛋白产物（如细胞核、核仁、高尔基体和细胞表面）的亚细胞定位来鉴定感兴趣的cDNA。利用这一策略，我们从含有造血祖细胞的胎鼠肝细胞中鉴定出一种新的锌指蛋白。我们还建立了一种新的信号序列陷阱方法，可以识别含有编码分泌和细胞表面信号序列的cDNA片段。信号序列陷阱最初是在京都大学开发的，由基因泰克公司的一个小组改进，已经建立了具有编码这些蛋白质的信号序列的分离cdna。在我们的方法中，将融合到血小板生成素（MPL）组成活性受体的细胞外缺失突变体的cDNA片段通过逆转录病毒感染引入il -3依赖性细胞，然后选择不依赖因子的克隆。我们的方法比以前发表的方法更快、更准确。我们已经鉴定出三种新的受体（一种I型细胞因子受体、一种TNF受体样分子和一种新型低密度脂蛋白受体相关蛋白）和几种新的SST-REX分泌分子，现在正在使用各种策略（包括敲除小鼠）表征这些分子。

We previously established a high-efficiency retrovirus-mediated expression cloning method. Using this system, we have now developed a novel expression cloning method in which cDNAs can be isolated based on the subcellular localization of their products. We make cDNAs using random hexamer and fuse them to 5' endo of the cDNA of green fluorescent protein(GFP)in the pMX retrovirus vector to construct a cDNA-GFP fusion retrovirus library. The derived retroviruses are then infected to NIH3T3 cells to identify a cDNA of interest based on the subcellular localization of its protein product such as nucleus, nucleoli, Golgi apparatus, and cell surface. Using this strategy, we have identified a novel zinc finger protein from fetal mouse liver cells that contain hematopoietic progenitor cells.We have also established a novel signal sequence trap method which can identify cDNA fragments containing signal sequence that encode secreted and cell-surface. Signal sequence trap was originally developed in Kyoto University, and was improved by a group in Genentech, isolate cDNAs with signal sequences that encode such proteins have been established. In our method termed, cDNA fragments fused to an extracellular deletion mutant of the constitutively active receptor for thrombopoietin(MPL)were introduced into IL-3-dependent cells via retrovirus infection followed by the selection of factor-independent clones. Our method is much quicker and more accurate than the previously published methods. We have identified three novel receptors(a type I cytokine receptor, a TNF receptor-like molecule, and a novel low-density lipoprotein receptor-related protein)and several novel secreted molecules by SST-REX, and are now characterizing these molecules using various strategies including knockout mice.