Exploiting orthologue diversity for systematic detection of gain-of-function phenotypes.
BACKGROUND
Systematic search for genes whose gain-of-function by exogenous expression confers an advantage in cell-based selective screenings is a powerful method for unbiased functional exploration of the genome, and has the potential to disclose new targets for cancer therapy. A major limit of this approach resides in the labor-intensive cloning of resistant cells, identification of the integrated genes and validation of their ability to confer a selective advantage. Moreover, the selection has to be drastic and genes conferring a limited advantage are typically missed.


RESULTS
We developed a new functional screening strategy based on transduction of mammalian cells of a given species with an expression library from another species, followed by one-shot quantitative tracing with DNA microarrays of all library-derived transcripts before and after selection. In this way, exogenous transcripts enriched after selection, and therefore likely to confer resistance, are readily detected. We transduced a retroviral cDNA expression library from mouse testis into human and canine cells, and optimized the use of commercial murine gene expression arrays for species-specific detection of library-derived transcripts. We then conducted a functional screening by growing library-transduced canine MDCK cells in suspension, to enrich for cDNAs conferring anchorage independence. Notably, these cells show partial resistance to loss of anchorage, and the selection can be of limited stringency, compromising approaches based on clonal selection or anyway requiring high stringency. Microarray analysis revealed reproducible enrichment after three weeks of growth on polyhema for seven genes, among which the Hras proto-oncogene and Sox5. When individually transduced into MDCK cells, Sox5 specifically promoted anchorage-independent growth, thereby confirming the validity and specificity of the approach.


CONCLUSIONS
The procedure described here brings substantial advantages to the field of expression cloning, being faster, more systematic and more sensitive. Indeed, this strategy allowed identification and validation of genes promoting anchorage-independent growth of epithelial cells under selection conditions not amenable to conventional expression cloning.
Martelli
Maria Luisa
ML
Laboratory of Functional Genomics, The Oncogenomics Center, Institute for Cancer Research and Treatment (IRCC), University of Turin Medical School, Str. Prov. 142, 10060 Candiolo, Italy. marialuisa.martelli@ircc.it
Isella
Claudio
C
Mira
Alessia
A
Fu
Limin
L
Cantarella
Daniela
D
Medico
Enzo
E
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D005240:Feasibility Studies; D015723:Gene Library; D014644:Genetic Variation; D006801:Humans; D008297:Male; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D000090063:Proto-Oncogene Mas; D015203:Reproducibility of Results; D055755:SOXD Transcription Factors; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013737:Testis / Q000378:metabolism; D014161:Transduction, Genetic
D004268:DNA-Binding Proteins; C000715470:MAS1 protein, human; D009687:Nuclear Proteins; D000090063:Proto-Oncogene Mas; D055755:SOXD Transcription Factors; C089186:Sox5 protein, mouse