(534b) Development of a Mammalian Cell-Based High Throughput Screening Method for the Directed Evolution of Genetic Switches

Selective control of endogenous gene expression has important applications in gene therapy, synthetic biology, and transgenic research. We describe a high throughput screening strategy in mammalian cells for the directed evolution of ligand-dependent gene switches capable of regulating gene expression. The strategy involves introducing a library of constructs into an EGFP HeLa reporter strain via protoplast fusion, followed by screening with FACS and plasmid recovery. This combines the ease of plasmid-based cloning with the realistic transcriptional milieu of mammalian cells. By performing screening in a mammalian system, unexpected host-specific results that can occur due to carrying out engineering in a model organism such as yeast are avoided. The screening method is being used to develop a new orthogonal ligand / gene switch pair consisting of the p65 transcriptional activator, a previously reported zinc finger domain targeting the vascular endothelial growth factor A (VEGF-A) promoter, and the estrogen receptor alpha ligand binding domain engineered to respond with sensitivity and specificity to a small-molecule compound. The construct design was optimized for inducible transcriptional activity by altering the order of various domains and the linker sequences in between. Expression of the gene switch in mammalian cells was verified by western blotting, and its effect on VEGF-A gene expression was monitored at both the mRNA and protein level. This and other engineered gene switches were tagged with EYFP and their localization and behavior in HeLa cells was visualized by fluorescence microscopy. The mammalian screening system will be used to pull out active clones from both saturation mutagenesis and error-prone PCR libraries, and should be useful for other projects where the engineered gene needs to be specifically expressed in mammalian cells.