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

To test the landing pad, we integrated a transcriptional unit that contains H2B fused to mAzamiGreen, driven by the CAG promoter (Fig. 4a) . Upon integration of the transfer vector into the hAAVS1 LP, we noted the decrease in mRuby2 expression together with expression of mAzamiGreen and tagBFP in most cells (Fig. 4b) , with a small fraction of cells showing no or incorrect integration of the transfer vector. Gratifyingly, clone #2, in which both wild type alleles of hAAVS1 locus were replaced by the landing pad construct, showed two populations as measured by fluorescence upon integration of the transfer vector. This suggested that these two populations had one or two copies that integrated into the genome, further demonstrating the efficiency of integrating into the landing pad ( Supplementary Fig. 3b ).