Issue 1, 2012

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

Abstract

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.

Graphical abstract: Quantitative assessment of Ras over-expression via shotgun deployment of vectors utilizing synthetic promoters

Supplementary files

Article information

Article type
Technical Innovation
Submitted
11 Aug 2011
Accepted
04 Nov 2011
First published
23 Nov 2011

Integr. Biol., 2012,4, 108-114

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