Flow cytometry of v-Abl transformed pre-B cells heterogeneous in ectopic expression levels reveals Ras dose-response.

Flow cytometry is a powerful tool for quantitative biology because it can perform single-cell analysis of large cell populations using multiple parameters. Results are often visualized as a two-dimensional scatter or contour plot. Because these plots can be relatively diffuse, it is not always straightforward to discern a relationship between measured parameters. We have demonstrated that quantitative trends can be fit to the single-cell data generated from a heterogeneous population. We engineered Abelson virus-transformed pre-B cells to express a broad range of oncogenic Ras levels. Instead of individual cultures with individual expression levels, a continuous range of levels was expressed by different cells in one heterogeneous culture. We then stained cells for downstream Erk phosphorylation to monitor MAPK signaling or employed an E2F-responsive genetic reporter to monitor cell-cycle activity. Subsequent analysis by flow cytometry and locally weighted scatterplot smoothing (LOWESS) revealed that increasing Ras oncogene expression led to increasing MAPK signaling. In contrast, E2F activity peaked at an optimal, intermediate level of Ras. To make this analytical method widely available to others, we have provided a software application that performs LOWESS on any two-parameter population data collected by flow cytometry.

Tetracycline-regulated expression implemented through transcriptional activation combined with proximal and distal repression.

Tetracycline-regulated expression systems are widely used to control ectopic gene expression in mammalian cells. However, background or "leaky" expression in the "off" state can limit applications that require control of expression at low levels. In this work we have engineered a tetracycline-regulated expression system with an improved range of control and lower background expression. To lower background expression without diminishing the controllable expression range, we designed a feed-forward scheme that repressed both expression of the gene of interest and the transcriptional activator. By using a tetracycline-responsive repressor that can modify chromatin and repress transcription over short and long distances, we were able to repress these two expression targets using a single tetracycline-responsive genetic element. This dual-targeting repressor/activation system demonstrated decreased background expression in its "off" state and a 25-fold range of expression in response to doxycycline. This study demonstrates that genetic circuits can be improved by leveraging trans-acting factors with long-range capabilities.

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

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.

A genetic reporter system to gauge cell proliferation rate.

In higher eukaryotes, E2F transcription factors often drive expression of genes necessary for the cell cycle, notably the G1/S phase transition. With conventional transcriptional reporter systems, expression of a reporter gene from an E2F-responsive promoter would allow one to identify the fraction of cells making this transition. Here, we have engineered an E2F-responsive genetic reporter system that outputs the proliferation rate. The system takes advantage of the long half-lives of fluorescent protein reporters and output signal normalization. By doing so, it converts dynamic pulses of E2F activity into an analog output proportional to the proliferation rate. Such a system should be useful for applications involving high-throughput drug or genetic screens, investigation of cellular environment, and biological engineering.

Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters.

UNLABELLED: The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11693-011-9089-0) contains supplementary material, which is available to authorized users.