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Koles K, Yeh AR, Rodal AA. Tissue-specific tagging of endogenous loci in Drosophila melanogaster. Biol Open. 2015;5(1):83-9doi: 10.1242/bio.016089.
Koles, K., Yeh, A. R., & Rodal, A. A. (2015). Tissue-specific tagging of endogenous loci in Drosophila melanogaster. Biology open, 5(1), 83-9. https://doi.org/10.1242/bio.016089
Koles, Kate, et al. "Tissue-specific tagging of endogenous loci in Drosophila melanogaster." Biology open vol. 5,1 (2015): 83-9. doi: https://doi.org/10.1242/bio.016089
Koles K, Yeh AR, Rodal AA. Tissue-specific tagging of endogenous loci in Drosophila melanogaster. Biol Open. 2015 Dec 23;5(1):83-9. doi: 10.1242/bio.016089. PMID: 26700726; PMCID: PMC4728311.
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Biol Open. 2015 Dec 23;5(1):83-9. doi: 10.1242/bio.016089.

Tissue-specific tagging of endogenous loci in Drosophila melanogaster.

Biology open

Kate Koles, Anna R Yeh, Avital A Rodal

Affiliations

  1. Department of Biology, Brandeis University, 415 South St, Waltham, MA 02454, USA [email protected] [email protected].
  2. Department of Biology, Brandeis University, 415 South St, Waltham, MA 02454, USA.

PMID: 26700726 PMCID: PMC4728311 DOI: 10.1242/bio.016089

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.

© 2016. Published by The Company of Biologists Ltd.

Keywords: CRISPR; Drosophila; Gene targeting; Golic+; OCRL; R recombinase/Rippase; Tissue-specific tagging; Vps35

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