Replication and episomal maintenance of Epstein-Barr virus-based vectors in mouse embryonal fibroblasts enable synthetic lethality screens - PubMed (original) (raw)

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PMID: 14617785

Replication and episomal maintenance of Epstein-Barr virus-based vectors in mouse embryonal fibroblasts enable synthetic lethality screens

Yulia Einav et al. Mol Cancer Ther. 2003 Nov.

Abstract

Recently, we demonstrated the establishment of chemical and genetic synthetic lethality screens in cultured human cells. Here, we report the establishment of this method in mouse embryonal fibroblasts (MEF). The method employs an immortalized mammalian cell line, deficient in a 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 green fluorescent protein (GFP)-based double-label fluorescence system. The crucial part in this endeavor has been the identification of a DNA replicon that could stably replicate in MEFs while under selection for survival and gets spontaneously lost relatively fast in the absence of such a pressure. Here, we show for the first time that EBV-based replicons but not polyoma virus-based ones can replicate and be stably maintained in MEFs. In the chemical screen, selective pressure imposed by synthetic lethal drugs prevented the spontaneous loss of the GFP-marked episome, enabling drug identification. Retention or spontaneous loss over time of the episomal survival plasmid could be sensitively detected in a large-scale blind test in the presence or absence of synthetic lethal chemicals, respectively. Establishing the synthetic lethality screen should thus permit high throughput screening for chemicals, which are synthetically lethal with any mouse mutant/knockout gene of interest. Moreover, it forms the basis for a genetic synthetic lethality screen in MEFs, an important new tool for mouse functional genomics.

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Replication and episomal maintenance of Epstein-Barr virus-based vectors in mouse embryonal fibroblasts enable synthetic lethality screens - PubMed (original) (raw)