Tagging endogenous loci for live-cell fluorescence imaging and molecule counting using ZFNs, TALENs, and Cas9.
The programmable ZFN, TALEN, and Cas9 nucleases allow genome editing of any cell line or organism. In this chapter, we describe methods to create gene fusions at endogenous loci in mammalian cells to express fluorescent fusions of proteins of interest at endogenous levels. The donor DNA, which includes the sequence encoding a fluorescent protein, is provided to the cell to repair a double-strand break induced by a nuclease. The engineered donor sequence is integrated by homology-directed repair into the genome in frame with the coding region of the gene of interest, resulting in expression of a fusion protein at physiological levels. We further describe techniques to study protein dynamics and numbers using the genome-edited cell lines. In contrast to cell lines stably overexpressing fusion proteins from modified cDNAs, genes encoding fluorescent proteins are targeted to the endogenous genetic locus, avoiding perturbation of alternative splicing and expression levels.
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA. Electronic address: dambournet@berkeley.edu.
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Dambournet
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
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SH
Hong
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
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Grassart
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
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DG
Drubin
D000818:Animals; D004247:DNA / Q000235:genetics; D018274:Electroporation; D004720:Endonucleases / Q000378:metabolism*; D018390:Gene Targeting; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D004720:Endonucleases
Donor plasmid; Endogenous; Fluorescence microscopy; GFP; Genome editing; Nuclease