Department of Molecular and Cell Biology, University of California, Berkeley 361 LSA, Berkeley, CA 94720-3200, USA.
Department of Molecular and Cell Biology, University of California, Berkeley 361 LSA, Berkeley, CA 94720-3200, USA
Development. 2015 Feb 1;142(3):597-606. doi: 10.1242/dev.114603.
Screens in mosaic Drosophila tissues that use chemical mutagenesis have identified many regulators of growth and patterning. Many of the mutant phenotypes observed were contingent upon the presence of both wild-type and mutant cells in the same tissue. More recently, large collections of RNAi lines or cDNAs expressed under Gal4/UAS control have been used to alter gene expression uniformly in specific tissues. However, these newer approaches are not easily combined with the efficient generation of genetic mosaics. The CoinFLP system described here enables mosaic screens in the context of gene knockdown or overexpression by automatically generating a reliable ratio of mutant to wild-type tissue in a developmentally controlled manner. CoinFLP-Gal4 generates mosaic tissues composed of clones of which only a subset expresses Gal4. CoinFLP-LexGAD/Gal4 generates tissues composed of clones that express either Gal4 or LexGAD, thus allowing the study of interactions between different types of genetically manipulated cells. By combining CoinFLP-LexGAD/Gal4 with the split-GFP system GRASP, boundaries between genetically distinct cell populations can be visualized at high resolution.
在使用化学诱变剂的马赛克果蝇组织中进行筛选,已经鉴定出许多生长和模式形成的调节因子。观察到的许多突变表型都取决于同一组织中野生型和突变型细胞的存在。最近,大量的 RNAi 系或在 Gal4/UAS 控制下表达的 cDNA 已被用于在特定组织中均匀改变基因表达。然而,这些较新的方法不容易与遗传马赛克的高效生成相结合。本文描述的 CoinFLP 系统通过以发育控制的方式自动生成可靠的突变型与野生型组织的比例,从而实现了基因敲低或过表达背景下的马赛克筛选。CoinFLP-Gal4 产生的组织由只有一部分表达 Gal4 的克隆组成。CoinFLP-LexGAD/Gal4 产生的组织由表达 Gal4 或 LexGAD 的克隆组成,从而允许研究不同类型的遗传操作细胞之间的相互作用。通过将 CoinFLP-LexGAD/Gal4 与分裂 GFP 系统 GRASP 相结合,可以高分辨率可视化遗传上不同的细胞群体之间的边界。