Patent ID: 6569623
Filing Date: 2003-05-27
Classification: C12N

Abstract:
A method for screening a molecule, wherein said molecule is a cDNA which has a gene-specific lethal property, said method comprising the steps of:a. transfecting a first reporter gene into a non-yeast eukaryotic cells having a genome comprising a gene of interest which carries a non-lethal mutation; b. selecting clones stably expressing said first reporter gene; c. introducing into said cells an episomal 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 plasmid is spontaneously lost from said cells; d. growing said cells in the presence of a selection compound which selects for said selectable marker; e. selecting cell clones stably expressing said second reporter gene and said functioning copy of said gene of interest; f. incorporating said cDNA into a vector containing a second selectable marker gene; g. transfecting cells with the vector resulting from step f); h. removing selection for the first selectable marker; i. selecting for cells expressing the second selectable marker gene; and j. determining episomal survival plasmid retention in cells, thus identifying a cDNA having a mutant gene-specific synthetic lethal property.