NATF (Native and Tissue-Specific Fluorescence): A Strategy for Bright, Tissue-Specific GFP Labeling of Native Proteins in Caenorhabditis elegans

Genetics: A Periodical Record of Investigations Bearing on Heredity and Variation

article

Siwei He¹  Andrea Cuentas-Condori²  David M. Miller¹ 

[1] Program in Neuroscience, Vanderbilt University, Nashville, Tennessee;Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37240

关键词: C. elegans;    cell specificity;    genome editing;    GFP;    NATF;    native expression;    protein localization;   

DOI  :  10.1534/genetics.119.302063

学科分类：医学（综合）

来源: Genetics Society of America

【 摘 要 】

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 of tissues that may obscure cell-specific localization. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy that combines genome editing and split-GFP to yield bright, cell-specific protein labeling. We use clustered regularly interspaced short palindromic repeats 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 the receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the Caenorhabditis elegans nervous system.

【 授权许可】

CC BY