Genetic synthetic lethality screen at the single gene level in cultured human cells.
Nucleic Acids Res
; 29(20): E100, 2001 Oct 15.
Article
en En
| MEDLINE
| ID: mdl-11600719
ABSTRACT
Recently, we demonstrated the feasibility of a chemical synthetic lethality screen in cultured human cells. We now demonstrate the principles for a genetic synthetic lethality screen. The technology employs both an immortalized human cell line deficient in the gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest, and the use of a novel GFP-based double-label fluorescence system. Dominant negative genetic suppressor elements (GSEs) are selected from an episomal library expressing short truncated sense and antisense cDNAs for a gene likely to be synthetic lethal with the gene of interest. Expression of these GSEs prevents spontaneous loss of the GFP-marked episomal survival plasmid, thus allowing FACS enrichment for cells retaining the survival plasmid (and the GSEs). The dominant negative nature of the GSEs was validated by the decreased resident enzymatic activity present in cells harboring the GSEs. Also, cells mutated in the gene of interest exhibit reduced survival upon GSE expression. The identification of synthetic lethal genes described here can shed light on functional genetic interactions between genes involved in normal cell metabolism and in disease.
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Genes Letales
Tipo de estudio:
Evaluation_studies
Límite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2001
Tipo del documento:
Article