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Series GSE11721

Query DataSets for GSE11721

Status

Public on Jun 10, 2008

Title

Exploiting orthologue diversity for systematic detection of gain-of-function phenotypes

Platform organism

Mus musculus

Sample organism

Canis lupus familiaris

Experiment type

Expression profiling by array

Summary

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.

Keywords: Xenoarray - a barcode-type microarray-based functional screening.

Overall design

7 samples: one control sample transduced with GFP, and six samples transduced with a mouse testis retroviral expression library, of which three selected for growth in the absence of anchorage. The cells are canine, the expression arrays are murine to trace the abundance of library-derived murine transcripts.

Contributor(s)

Medico E, Isella C

Citation(s)

18510758

Submission date

Jun 09, 2008

Last update date

Mar 19, 2012

Contact name

Enzo Medico

E-mail(s)

enzo.medico@ircc.it