Ramot University Authority for Applied Research and Industrial Development Limited

Arnold Simons

Dan Canaani

Iris Dotan

Naomi Dafni

Yoram Oron

A method for screening molecule which have a synthetic lethal property when in combination with a gene of interest carrying a non-lethal mutation, said method comprising the steps of: transfecting a first reporter gene into mammalian cells having a genome comprising a gene of interest which carries a non-lethal mutation, or a genome which is null of said gene of interest; selecting clones stably expressing said fist reporter gene; introducing into said cells a survival plasmid comprising a functioning copy of said gene of interest, a second reporter gene, selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the plasmid within said cells, wherein said survivsal plasmid is autonomously replicating and spontaneously lost from said cells; growing said cells in the presence of a selection compound which selects for said selectable marker; selecting cell clones stably expressing said second reporter gene and said functioning copy of said gene of interest; removing selection for the selectable marker, and adding molecules destined for screening of their ability to impose selective pressure enforcing retention of the unstable survival plasmid, determining survival plasmid retention in cells, thus identifying a molecule having a synthetic lethal property when in combination with non lethal mutated gene of interest.

Genetic screening methods

A screening method which applies the principle of synthetic lethality to a gene of interest in non-yeast eukaryotic cells. The method may be used to screen either a chemical library in order to identify a molecule having a gene-specific lethal property in the cells, or to screen a group of DNA molecules in order to identify among them one or more modulators of gene function which are synergistically lethal to the cells together with an incapacitated gene of interest. Also described are episomal survival plasmids and kits which may be used with the method.

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Carcur, Israel

Arnold Simons