Analysis and optimization of gene expression using synthetic promoters

Applications

• Tune gene expression in synthetic biology devices, genetic circuits, and engineered cells.
• Identify optimal individual gene and multi-gene expression levels for generating specific cell behaviors and phenotypes, including proliferation, apoptosis, senescence, tumorigenesis, metatastasis, cell differentiation, and generation of stem cells from terminally differentiated somatic cells.
• Generate cell populations that can be used to screen for therapeutics that abrogate synergy between genes or expressed factors. The invention can be used to generate dose-responses for genes of interest.

Advantages

• High compatibility with existing expression systems - The promoters have been generated from constitutive promoters already commonly used in practice.
• Efficient - Use of viral expression vectors allows for efficient gene delivery and makes high-throughput gene transduction possible.
• Fast with high resolution - The method is able to generate gene expression dose-response curves with higher resolution in a faster manner than other methods.

Publications

• Ferreira, J.P., Peacock, R.W., Lawhorn, I.E., and Wang, C.L. (2011). "Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters." Syst Synth Biol (in press).
• Ferreira, J.P., W., Lawhorn, I.E., Peacock, R. and Wang, C.L. (2012) "Quantitative assessment of Ras over-expression via shotgun deployment of vectors utilizing synthetic promoters." Integr. Biol. (Camb).
• Peacock, R.W., Lawhorn, I.E., Ferreira, J.P. and Wang, C.L. (2012). "Flow cytometry of v-Abl transformed pre-B cells heterogeneous in ectopic expression levels reveals Ras dose-response." J Immunol Methods 384, 177-183.

Stage of Research

This method has been tested, demonstrating that it can be used to tune and optimize gene expression levels in mammalian cells.

As an example of this method, named SPLAT (Synthetic Promoter Library Aggregate Transduction), the researchers determined the dose-dependent effects of Ras expression. Dose-response curves indicated that a single-copy level of oncogenic Ras (HRAS G12V) generated maximal imatinib resistance and activated MAPK as effectively as six-fold amplification of wild-type Ras higher expression levels led to decreasing proliferation rates.