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 next employed the PiggyBac transposon system to achieve high copy integration of the ZEBOV-4cis construct for a robust functional minigenome reporter readout. HEK293T cells were co-transfected with PiggyBac transposon and the PiggyBac donor construct (MTK043-ZEBOV-4cis) that contains a hygromycin marker cassette, followed by selection with hygromycin for 7 days (Fig. 7c) . This resulted in 2 independent stable populations of HEK293T cells expressing ZEBOV RNP proteins, as determined by eGFP minigenome reporter signal ( Supplementary Fig. 5c ). Clonal cell lines were isolated and expanded from these 2 stable . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/506188 doi: bioRxiv preprint populations in an additional 1-2 weeks. This workflow simplified and speeded the generation of stable EBOV RNP cell lines compared to the previously reported system 27 .