Quantitative assessment of Ras over-expression viashotgun deployment of vectors utilizing synthetic promoters

• Published in: Integrative biology (Cambridge) 2012-01, Vol.4 (1), p.108-114
• Main Authors: Ferreira, Joshua P, Lawhorn, Ingrid EB, Peacock, Ryan WS, Wang, Clifford L
• Format: Article
• Language: English
• Subjects: Dose-response effects; Expression vectors; Flow cytometry; Gene expression; Imatinib; MAP kinase; Overexpression; Promoters; Ras protein; Signal transduction
• 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.