Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125;
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
Proc Natl Acad Sci U S A. 2018 Apr 10;115(15):3900-3905. doi: 10.1073/pnas.1720063115. Epub 2018 Mar 26.
Bipartite expression systems, such as the GAL4-UAS system, allow fine manipulation of gene expression and are powerful tools for interrogating gene function. Recently, we established cGAL, a GAL4-based bipartite expression system for transgene control in , where a single promoter dictates the expression pattern of a cGAL driver, which then binds target upstream activation sequences to drive expression of a downstream effector gene. Here, we report a split strategy for cGAL using the split intein gp41-1 for intersectional control of transgene expression. Split inteins are protein domains that associate, self-excise, and covalently ligate their flanking peptides together. We split the DNA binding domain and transcriptional activation domain of cGAL and fused them to the N terminal of gp41-1-N-intein and the C terminal of gp41-1-C-intein, respectively. In cells where both halves of cGAL are expressed, a functional cGAL driver is reconstituted via intein-mediated protein splicing. This reconstitution allows expression of the driver to be dictated by two promoters for refined spatial control or spatiotemporal control of transgene expression. We apply the split cGAL system to genetically access the single pair of MC neurons (previously inaccessible with a single promoter), and reveal an important role of protein kinase A in rhythmic pharyngeal pumping in Thus, the split cGAL system gives researchers a greater degree of spatiotemporal control over transgene expression, and will be a valuable genetic tool in for dissecting gene function with finer cell-specific resolution.
双组份表达系统，如 GAL4-UAS 系统，允许对基因表达进行精细操作，是研究基因功能的有力工具。最近，我们建立了 cGAL，这是一个基于 GAL4 的双组份表达系统，用于 中转基因的控制，其中单个启动子决定 cGAL 驱动子的表达模式，然后结合靶上游激活序列驱动下游效应基因的表达。在这里，我们报告了一种使用分裂整合蛋白 gp41-1 的 cGAL 分裂策略，用于转基因表达的交叉控制。分裂整合蛋白是一种蛋白质结构域，它们会结合、自我切除，并将其侧翼肽共价连接在一起。我们将 cGAL 的 DNA 结合域和转录激活域分裂，并分别将其融合到 gp41-1-N-整合蛋白的 N 端和 gp41-1-C-整合蛋白的 C 端。在表达 cGAL 两半的细胞中，通过整合酶介导的蛋白剪接重新构成功能性 cGAL 驱动子。这种重建允许通过两个启动子来控制驱动子的表达，从而实现更精细的空间控制或转基因表达的时空控制。我们应用分裂 cGAL 系统来遗传访问单个 MC 神经元（以前无法通过单个启动子访问），并揭示了蛋白激酶 A 在 节律性咽泵中的重要作用。因此，分裂 cGAL 系统为研究人员提供了对转基因表达更高程度的时空控制，并将成为 中用于以更精细的细胞特异性分辨率解析基因功能的有价值的遗传工具。
