Protein-protein interaction panel using mouse full-length cDNAs

Citation
H. Suzuki et al., Protein-protein interaction panel using mouse full-length cDNAs, GENOME RES, 11(10), 2001, pp. 1758-1765
Citations number
29
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Molecular Biology & Genetics
Journal title
GENOME RESEARCH
ISSN journal
1088-9051 → ACNP
Volume
11
Issue
10
Year of publication
2001
Pages
1758 - 1765
Database
ISI
SICI code
1088-9051(200110)11:10<1758:PIPUMF>2.0.ZU;2-F
Abstract
We have developed a novel assay system for systematic analysis of protein-p rotein interactions (PPIs) that is characteristic of a PCR-mediated rapid s ample preparation and a high-throughput assay system based on the mammalian two-hybrid method. Using gene-specific primers, we successfully constructe d the assay samples by two rounds of PCR with up to 3.6 kb from the first-r ound PCR fragments. In the assay system, we designed all the steps to be pe rformed by adding only samples, reagents, and cells into 384-well assay pla tes using two types of semiautomatic multiple dispensers. The system enable d us examine more than 20,000 assay wells per day. We detected 145 interact ions in our pilot study using 3500 samples derived from mouse full-length e nriched cDNAs. Analysis of the interaction data showed both several signifi cant interaction clusters and predicted functions of a few uncharacterized proteins. In combination with our comprehensive mouse full-length cDNA clon e bank covering a large part of the whole genes, our high-throughput assay system will discover many interactions to facilitate understanding of the f unction of uncharacterized proteins and the molecular mechanism of crucial biological processes, and also enable completion of a rough draft of the en tire PPI panel in certain cell types or tissues of mouse within a short tim e.