Selection-subtraction approach (SSA): a universal genetic screening technique that enables negative selection.
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.
Singhi
Aatur D
AD
Department of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Kondratov
Roman V
RV
Neznanov
Nickolay
N
Chernov
Mikhail V
MV
Gudkov
Andrei V
AV
D001483:Base Sequence; D030561:Databases, Nucleic Acid; D015723:Gene Library; D005820:Genetic Testing / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011089:Polydeoxyribonucleotides / Q000737:chemistry*; D011467:Prostate; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D016372:RNA, Antisense / Q000235:genetics; D015183:Restriction Mapping; D012641:Selection, Genetic
D011089:Polydeoxyribonucleotides; D011506:Proteins; D016372:RNA, Antisense