Quantitative assessment of Ras over-expression via shotgun deployment of vectors utilizing synthetic promotersElectronic supplementary information (ESI) available. See DOI: 10.1039/c1ib00082a

• Main Authors: Ferreira, Joshua P, Lawhorn, Ingrid E. B, Peacock, Ryan W. S, Wang, Clifford L
• Format: Article
• Language: English
• ISSN: 1757-9694; 1757-9708
• DOI: 10.1039/c1ib00082a

We sought to characterize and compare wild-type and oncogenic Ras over-expression. Because different levels of Ras over-expression can have different effects on cell phenotype, it was important to evaluate a wide range of expression. Different expression levels were achieved by using retroviral vectors equipped with different strength promoters. Cells were shotgun transduced with a mixture of these vectors to generate heterogeneous populations exhibiting a range of expression levels. We used flow cytometry to analyze the populations and generate high-resolution, nearly continuous Ras dose-response curves. These efforts revealed that a single-copy level of oncogenic Ras generated maximal imatinib resistance and activated MAPK pathway signaling as effectively as six-fold amplification of wild-type Ras. Although further increased expression lead to even greater signal transduction, this increased expression had minimal or decreasing effects on the proliferation rate. In addition, this study introduces a general method to quantify genetic dose-response relationships and identify gene expression ranges that produce an optimized phenotypic response. Cells were transduced with Ras transgenes expressed from various synthetic promoters. High resolution dose-response curves relating expression and phenotype were achieved through analysis of cell heterogeneity.