A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

João Pedro Fonseca; Alain R. Bonny; G. Renuka Kumar; Andrew H. Ng; Jason Town; Qiu Chang Wu; Elham Aslankoohi; Susan Y. Chen; Patrick Harrigan; Lindsey C. Osimiri; Amy L. Kistler; Hana El-Samad

We characterized the resulting EBOV RNP stable cell lines via quantitative minigenome reporter assays, protein expression and localization studies, and analysis of susceptibility to previously described small molecule inhibitors of ZEBOV RNP minigenome activity. ZEBOV-4cis stable populations and clonal cells yielded comparable nLuc minigenome reporter activity (Fig. 7d ). ZEBOV viral proteins in both the stable and clonal cell populations were readily detectable with NP and VP35 specific antibodies, and showed expression levels similar to transiently transfected ZEBOV-4cis (Fig. 7e) . Additionally, based on the presence of 2A peptide tag that remains at the C-terminus of each of these proteins following "self-cleavage" due to ribosome skipping, NP, VP35, and VP30 were each detectable with an antibody raised against the 2A peptide (Fig. 7e) . Larger multi-ORF fusion protein products were not readily detected with the 2A peptide antibodies, indicating efficient cleavage occurred at each of the P2A sites encoded within the ZEBOV-4cis construct. This demonstrates the added benefit that the P2A parts in the MTK provide a potential "universal" method to detect protein expression from multicistronic constructs. This is especially useful for analysis of novel emerging viruses or other proteins where specific antibodies are not available.