Since establishing the Molecular and Cell Biology group in August 2008, lab personnel have been hired, a first generation version of the robotic screening platform brought into service, and significant progress made in preparation for running our first RNAI screens in monocyte/macrophage cell lines. Assay development: We plan to run screens using assays with both a microscopy-based 'high content'single cell readout, and also bioluminescence-based population assays using luciferase reporters driven by inflammatory gene promoters. We have designed and constructed dual promoter lentiviral vectors that permit the expression of two genes from a single virus. This has led to the creation of a RAW264.7 mouse macrophage cell line expressing two fluorescent biosensors for high content screening. The first readout expresses a GFP fusion with the RelA NFkB transcription factor driven by its endogenous promoter. This protein partitions to the cytoplasm in unstimulated cells, and translocates to the nucleus in response to a wide range of ligands that promote macrophage activation. Use of the endogenous RelA promoter facilitates a more accurate reproduction of the oscillatory cytosol/nuclear translocation previously observed with endogenous RelA. The second biosensor uses the murine TNF alpha promoter to drive expression of the red fluorescent protein mCherry, fused to a destabilizing PEST sequence that reduces the protein's half-life in the cell. This latter modification provides a more dynamic readout of TNF alpha promoter activity in kinetic experiments. We have established a clonal cell line exhibiting robust RelA translocation in the first hour, followed by a significant increase in TNF alpha promoter-driven mCherry expression after 12 hours in response to a wide range of Toll-like receptor (TLR) ligands. We have used a similar strategy to create a THP1 human monocyte cell line expressing firefly luciferase driven by the human TNF alpha promoter and also renilla luciferase driven by the ubiquitin promoter. The constitutive renilla expression provides a valuable normalization factor for cell number variability in a population-based readout. Thus, the firefly/renilla ratio in this cell line after TLR stimulation provides a measure of TNF alpha promoter activity, and we see a significant and reproducible increase in response to various TLR ligands. Interestingly, this reporter responds more quickly to LPS in this THP1 cell line (2-4 hr) than does mCherry in the RAW cell line (12-16 hr). Although the higher sensitivity of the luciferase assay may contribute to this, this behavior likely reflects some difference in the response kinetics of the human and mouse TNF alpha promoters, a finding that may have clinical relevance. Optimization of siRNA delivery: Effective delivery of siRNA into hematopoietic cells remains a significant obstacle to the implementation of effective siRNA screens. It is important to establish a reproducible method that can achieve >80% knockdown (KD) of target genes in order to avoid a high frequency of false negatives in genome-wide screens. We have taken advantage of the fact that the creation of the RAW and THP1 reporter cell lines described above provide convenient control siRNA targets in the reporters they express. The ability to assay GFP and renilla luciferase KD in the RAW and THP1 lines respectively provides two advantages. First, it allows us to assay for protein rather than mRNA KD (the most common validation method for siRNA delivery), providing a more direct measure of the required endpoint needed for an effective screen. Second, the ability to run both assays in 384-well format allows for a more extensive matrix of experimental conditions, which improves the chance of identifying an optimal delivery protocol. Using such an approach, we are in the final stages of validating highly efficient lipid-based transfection protocols in 384-well format for both of the described RAW and THP1 cell lines. Robotic workflows: Robotic equipment was purchased previously for high content screens: a cytomat incubator with capacity for >150 microplates, a Tecan evo150 liquid handler and a BD pathway bioimager, with robotic integration of the three machines through a Thermo catalyst express robotic arm. We have established robotic workflows for screens in 384-well plates using the RAW cell line described above. This will permit the recording of both the RelA-GFP and TNF alpha promoter-mCherry readouts from the same live cells over a 12+ hr time course. We predict that ability to score multiple phenotypes from the same cells, and to compare both single cell and population data in the same RNAi screen will be a valuable feature of the described assay platform. Once we have completed controls for assay reproducibility we expect to run preliminary screens within the next few months with siRNAs targeting TLR signaling pathway genes previously identified by other laboratories using murine in vivo mutagenesis methods. We have purchased whole genome siRNA libraries, and envision running large-scale screens by early next year. In addition, we are collaborating with Scott Martin, Natasha Caplen and Chris Austin in their efforts to establish a trans-NIH RNAi screening group at the National Chemical Genomics Center. We will provide one or both of the cell lines described above for the NCGC RNAi group to run parallel screens using their robotic infrastructure. This will provide an important validation of protocols in both groups, as well as increasing confidence in screen hits that overlap in each dataset.
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