Screens using RNAi and cDNA expression as surrogates for genetics in mammalian tissue culture cells.
We have developed methods for the automation of transfection-grade DNA preparation, high-throughput retroviral preparation, and highly parallel phenotypic screens to establish approaches that will allow investigators to examine in an unbiased manner the roles of proteins in mammalian cells. These methods have been used to raise or lower the levels of individual kinases in individual micro-well cultures either by cDNA or short hairpin RNA expression and will allow investigators to treat mammalian cells in culture in manners that are analogous to genetic screens in yeast. Our proof-of-principle experiments have been performed in human cells using repositories that represent over 75% of the protein, nucleotide, carbohydrate, lipid, and amino acid kinases in the human genome. These initial experiments have demonstrated the feasibility of two general types of screens. We have performed phenotypic screens to identify proteins with specific roles in a chosen function and genetic interaction screens to establish epistatic relations between different proteins. The results suggest that any phenotype that can be scored by a robust assay in tissue culture is amenable to these types of screens and that interactions between mammalian proteins can be established. These results point to the near-term goal of establishing comprehensive, unbiased screens that will allow queries on the roles of all human proteins.
Pearlberg
J
J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Degot
S
S
Endege
W
W
Park
J
J
Davies
J
J
Gelfand
E
E
Sawyer
J
J
Conery
A
A
Doench
J
J
Li
W
W
Gonzalez
L
L
Boyce
F M
FM
Brizuela
L
L
Labaer
J
J
Grueneberg
D
D
Harlow
E
E
D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology / Q000235:genetics; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D005820:Genetic Testing; D023281:Genomics; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology / Q000235:genetics; D010641:Phenotype; D010770:Phosphotransferases / Q000235:genetics; D034622:RNA Interference; D012190:Retroviridae / Q000235:genetics; D013489:Suppression, Genetic; D014162:Transfection
D018076:DNA, Complementary; D010770:Phosphotransferases