(74b) Development of a Constitutive Promoter Library for Optimization of Cell Lines

With the tools that are readily available, studying the effects of gene expression typically means knocking-out expression of the gene of interest or over-expression -- possibly at non-physiological levels. To investigate a broader range of gene expression, we have developed a library of constitutive promoters capable of driving transcription at varying levels. Our library allows for interrogation of dosages between the extremes currently used and may more closely reflect expression levels a cell normally experiences. Inducible systems are leaky at the low end and not necessarily linear over all concentrations of inducer; furthermore, studying multiple genes is limited by the number of orthogonal inducible systems available. Expression from the constitutive promoters results in defined expression profiles and multiple genes of interest can be tied to expression of multiple fluorescent proteins, vastly increasing the number of genes analyzed in combination. Using this library we have generated profiles correlating expression levels of the activated forms of the proto-oncogenes c-MYC and H-RAS to proliferation rates in various cell lines.

Results from these individual, single gene experiments give insight into optimal expression patterns for a particular phenotype, here proliferation. Establishing cell lines requiring low to no serum results in lower costs and greater uniformity in cultures. To this end, we have utilized our promoter library system in mouse pre-B and CHO cells and correlated expression of c-MYC and H-RAS with proliferation at varying serum concentrations. We have also studied the effects that varying expression of the anti-apoptosis gene BCL2 has on cell survival at these different serum levels. The advantage of our system will be in combining multiple genes at varying dosages to optimize proliferation rate and resistance to apoptosis.