A putative approach for cloning 'silent' genes using retroviral vectors.
An alternative approach is described for cloning 'silent' or poorly expressed genes that might be activated as a result of proviral DNA integration. Generally, proviral DNA integrates randomly, in a single copy per cell, although some preferred integration sites exist. Thus, each cell infected by a retroviral vector should represent a different clone. These clones would have different phenotypes depending on the site of proviral DNA integration. Those expressing a desired phenotype would be screened for by using an assay system that depends on the gene of interest. The 5' and 3' flanking cellular DNA sequences, responsible for the observed phenotype in such mutants, could be cloned. Screening of these clones should be relatively simple due to the presence of a selectable marker in the proviral DNA. These sequences would then be used to isolate the wild type (wt) copy of the insertionally activated gene. Packaging retroviral vectors could lead to insertional activation of cellular genes and packaging of insertionally activated RNAs into vector particles. RNA extracted from the particles released into the culture medium would greatly facilitate cDNA cloning of 'silent' genes activated as a result of proviral DNA integration.
Joshi
S
S
Department of Microbiology, University of Toronto, Ontario, Canada.
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D016254:Mutagenesis, Insertional; D012190:Retroviridae / Q000235:genetics*
D004247:DNA