A rapid genetic screening system for identifying gene-specific suppression constructs for use in human cells.
We describe a rapid cell-based genetic screen using fission yeast for identifying efficient gene suppression constructs (GSCs) from large libraries (10(5)) for any target sequence for use in human cells. In this system, target sequences are fused to the 5' end of the lacZ reporter gene and expressed in yeast. Random fragment expression libraries derived from the target sequence are screened in the fusion gene-expressing strain using the lacZ gene-encoded colony color phenotype. We demonstrate the utility of this screening assay by identifying a range of different GSCs for the fission yeast ura4 gene and human c-myc and Chk1 sequences, including rare efficient suppressors. GSCs specific for c-myc were shown to regulate expression of both a c-myc-lacZ fusion gene and the endogenous c-myc gene in human cells.
Arndt
G M
GM
Johnson & Johnson Research, The Australian Technology Park, Level 4, 1 Central Avenue, Eveleigh, NSW 1430, Sydney, Australia. garndt@medau.jnj.com
Patrikakis
M
M
Atkins
D
D
D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D005656:Fungal Proteins / Q000235:genetics; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D015723:Gene Library; D016259:Genes, myc; D006801:Humans; D007763:Lac Operon; D010957:Plasmids; D011494:Protein Kinases / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins
D005656:Fungal Proteins; D029702:Schizosaccharomyces pombe Proteins; C400810:ura4 protein, S pombe; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe