[Protein knockout (as opposed to mere knockdown)]
>>Published online before print October 30, 2003
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.2233012100
Genetics
Exploring the functional complexity of cellular proteins by protein knockout
Jianxuan Zhang *, Ning Zheng , and Pengbo Zhou *
*Department of Pathology and Laboratory Medicine, Graduate Program in Molecular Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021; and Department of Pharmacology, University of Washington, Seattle, WA 98195
Edited by Mark Ptashne, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved September 3, 2003 (received for review May 19, 2003)
Comprehensive dissection of protein functions entails more complicated manipulations than simply eliminating the protein of interest. Established knockdown technologies, such as RNA interference, antisense oligodeoxynucleotides, or ribozymes, are limited for specific applications such as modulating protein levels or specific targeting of a posttranslationally modified subpopulation. Here we show that the engineered Skp1, Cullin 1, and F-box-containing TrCP substrate receptor ubiquitin-proteolytic system, designated protein knockout, could achieve not only total elimination but also rapid and systematic reduction of a given cellular protein. Stable expression of a single engineered TrCP demonstrated simultaneous and sustained degradation of the entire retinoblastoma family proteins. Furthermore, the engineered TrCP was capable of selecting hypo- but not hyperphosphorylated forms of retinoblastoma for degradation. The engineered TrCP has been extensively modified to increase its specificity in substrate selection. This optimized protein-knockout system offers a powerful and versatile proteomic tool to dissect diverse functional properties of cellular proteins in somatic cells.<<
Worth watching this technology, IMO.
Cheers, Tuck |
