title: Generation of a fluorescently labeled endogenous protein library in living human cells.
abstract: We present a protocol to tag proteins expressed from their endogenous chromosomal locations in individual mammalian cells using central dogma tagging. The protocol can be used to build libraries of cell clones, each expressing one endogenous protein tagged with a fluorophore such as the yellow fluorescent protein. Each round of library generation produces 100-200 cell clones and takes about 1 month. The protocol integrates procedures for high-throughput single-cell cloning using flow cytometry, high-throughput cDNA generation and 3' rapid amplification of cDNA ends, semi-automatic protein localization screening using fluorescent microscopy and freezing cells in 96-well format.
authors.affiliation: Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel. sigal@caltech.edu
authors.forename: Alex
authors.initials: A
authors.lastname: Sigal
authors.forename: Tamar
authors.initials: T
authors.lastname: Danon
authors.forename: Ariel
authors.initials: A
authors.lastname: Cohen
authors.forename: Ron
authors.initials: R
authors.lastname: Milo
authors.forename: Naama
authors.initials: N
authors.lastname: Geva-Zatorsky
authors.forename: Gila
authors.initials: G
authors.lastname: Lustig
authors.forename: Yuvalal
authors.initials: Y
authors.lastname: Liron
authors.forename: Uri
authors.initials: U
authors.lastname: Alon
authors.forename: Natalie
authors.initials: N
authors.lastname: Perzov
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry*; D045744:Cell Line, Tumor; D002470:Cell Survival; D005453:Fluorescence; D005786:Gene Expression Regulation; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis* / Q000737:chemistry*; D019151:Peptide Library; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D019151:Peptide Library; C054562:yellow fluorescent protein, Bacteria