NATF (Native and Tissue-Specific Fluorescence): A Strategy for Bright, Tissue-Specific GFP Labeling of Native Proteins in Caenorhabditis elegans
Year: 2019
Type: article
Abstract: ABSTRACT GFP labeling by genome editing can reveal the authentic location of a native protein but is frequently hampered by weak GFP signals and broad expression across a range cell types in multicellular animals. To overcome these problems, we engineered a N ative A nd T issue-specific Fluorescence (NATF) strategy which combines CRISPR/Cas-9 and split-GFP to yield bright, cell-specific protein labeling. We use CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 β-strand ( gfp11 x7 ) at the genomic locus of each target protein. The resultant gfp11 x7 knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment ( gfp1 - 10 ) in specific cell types thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein, OIG-1, and demarcates a receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the C. elegans nervous system.
Source: Genetics
Institutions: Vanderbilt University, Vanderbilt University Medical Center
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2928476716