Conflicts with diarrheal pathogens trigger rapid evolution of an intestinal signaling axis

Clayton M. Carey; Sarah E. Apple; Zoe A. Hilbert; Michael S. Kay; Nels C. Elde

Lentivirus generation and cloning GC-C sequences were downloaded from GenBank based on whole genome sequences from each species tested. Each C-terminally HA tagged GC-C variant was synthesized via gene synthesis (Life Technologies). GC-C was then cloned into the lentiviral transfer vector pUltra (Addgene #24129) between the XbaI and BamHI restriction sites by Gibson Assembly, in frame with GFP and the T2A linker sequence. To generate lentiviral particles, 10 cm dishes were seeded with 3 x 10 6 293T cells 24 hours prior to transfection. Cells were then transfected with 7.6 µg pUltra-GC-C, 7.6 µg psPAX2 packaging plasmid (Addgene # 12260), and 3.8 µg pMD2.G envelope plasmid (Addgene # 12259) with 56 µl FuGene HD transfection reagent according to the manufacturer's specifications. Media was replaced 24 hours post-transfection and replaced with 10 mL media. Viral supernatants were collected 48 hours-post transfection and passed through a 0.4 µm followed by overnight incubation with 1X PEG-IT solution (System Biosciences) at 4°C. Precipitated viral particles were centrifuged at 1500xg for 30 min at 4°C and resuspended in PBS at a final volume of 500 µl before storage at -80°C.