Open AccessSelection–subtraction approach (SSA): A universal genetic screening technique that enables negative selection
Author(s) -
Aatur D. Singhi,
Roman V. Kondratov,
Nickolay Neznanov,
Mikhail Chernov,
Andrei V. Gudkov
Publication year - 2004
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.0403080101
Subject(s) - biology , mutant , computational biology , selection (genetic algorithm) , gene , genetics , genomic library , peptide library , microbiology and biotechnology , peptide sequence , artificial intelligence , computer science
Screening of expression libraries for bioactive clones that modulate the growth of mammalian cells has been limited largely to positive selections incapable of revealing growth suppressive or lethal genetic elements. We have developed a technique, selection-subtraction approach (SSA), that allows growth-modulating clones to be isolated based on alterations in their relative abundance in growing cell populations that have been transduced with an expression library. SSA utilizes tagged retroviral libraries in bacteriophage lambda vectors (retrophages). Nylon prints from retrophage libraries are used to determine the relative abundance of tags in library-transduced cells to identify biological activity of individual clones. Applications of SSA for gene discovery, target discovery, and generation of mutant proteins have been demonstrated, by using p53 and ataxia telangiectasia mutated (ATM) as models to isolate growth inhibitory proteins, peptides and antisense RNAs, and temperature-sensitive mutant proteins.