Generation of a fluorescently labeled endogenous protein library in living human cells.
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.
Sigal
Alex
A
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel. sigal@caltech.edu
Danon
Tamar
T
Cohen
Ariel
A
Milo
Ron
R
Geva-Zatorsky
Naama
N
Lustig
Gila
G
Liron
Yuvalal
Y
Alon
Uri
U
Perzov
Natalie
N
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*
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D019151:Peptide Library; C054562:yellow fluorescent protein, Bacteria