Tissue-specific tagging of endogenous loci in Drosophila melanogaster.
Tissue-specific tagging of endogenous loci in Drosophila melanogaster.
复制标题
果蝇中内源性基因座的组织特异性标记。
DOI：
10.1242/bio.016089
复制
发表时间：
2015-12-23
期刊：
影响因子：
2.4
通讯作者：
Rodal AA
中科院分区：
文献类型：
--
作者：
Koles K;Yeh AR;Rodal AA
荧光蛋白标签已经彻底改变了细胞和发育生物学，并与二元表达系统相结合，它们使蛋白质功能的各种组织特异性研究成为可能。然而，这些二元表达系统通常不能概括内源性蛋白质表达水平、定位、结合配偶体和/或基因表达的发育窗口。为了解决这些局限性，我们开发了一种称为T-STEP（内源性蛋白质的组织特异性标记）的方法，该方法允许以组织特异性方式标记内源性基因座。T-STEP使用有效的CRISPR/Cas9增强的基因靶向和组织特异性重组酶介导的标签交换的组合来在时间和空间上标记内源性蛋白质。我们采用这种方法在包括神经元、神经胶质细胞、肌肉和血细胞在内的多种果蝇组织中标记GFP OCRL（内吞途径中的磷酸肌醇-5-磷酸酶）和Vps 35（内体逆转录聚合物中与帕金森病相关的成分）。内源性蛋白质的选择性标记首次允许复杂组织中的细胞类型特异性活成像和蛋白质组学。总结：在这里，我们描述了一个容易适用的方法，允许C-末端蛋白标记在内源性基因座在组织或细胞类型特异性的方式在果蝇。
Fluorescent protein tags have revolutionized cell and developmental biology, and in combination with binary expression systems they enable diverse tissue-specific studies of protein function. However these binary expression systems often do not recapitulate endogenous protein expression levels, localization, binding partners and/or developmental windows of gene expression. To address these limitations, we have developed a method called T-STEP (tissue-specific tagging of endogenous proteins) that allows endogenous loci to be tagged in a tissue specific manner. T-STEP uses a combination of efficient CRISPR/Cas9-enhanced gene targeting and tissue-specific recombinase-mediated tag swapping to temporally and spatially label endogenous proteins. We have employed this method to GFP tag OCRL (a phosphoinositide-5-phosphatase in the endocytic pathway) and Vps35 (a Parkinson's disease-implicated component of the endosomal retromer complex) in diverse Drosophila tissues including neurons, glia, muscles and hemocytes. Selective tagging of endogenous proteins allows, for the first time, cell type-specific live imaging and proteomics in complex tissues. Summary: Here we describe an easily applicable method that allows C-terminal protein tagging at endogenous loci in a tissue- or cell type-specific manner in Drosophila melanogaster.