title: Tissue-specific tagging of endogenous loci in Drosophila melanogaster.
abstract: 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.
authors.affiliation: Department of Biology, Brandeis University, 415 South St, Waltham, MA 02454, USA k.koles@gmail.com arodal@brandeis.edu.
authors.forename: Kate
authors.initials: K
authors.lastname: Koles
authors.affiliation: Department of Biology, Brandeis University, 415 South St, Waltham, MA 02454, USA.
authors.forename: Anna R
authors.initials: AR
authors.lastname: Yeh
authors.affiliation: Department of Biology, Brandeis University, 415 South St, Waltham, MA 02454, USA k.koles@gmail.com arodal@brandeis.edu.
authors.forename: Avital A
authors.initials: AA
authors.lastname: Rodal
keywords: CRISPR; Drosophila; Gene targeting; Golic+; OCRL; R recombinase/Rippase; Tissue-specific tagging; Vps35