Pcdh cmv mcs ef1a gfp neo

The N-terminal mCherry-IRGB10 fusion construct was constructed using the NEBuilder HiFi DNA assembly kit (E2621S, New England BioLabs) according to the manufacturer’s protocol. PCR products were generated for mCherry (5’-AGAAGATTCTAGAGCTAGCGATGGTGAGCAAGGGCGAG-3’) and (5’-CTTGTACAGCTCGTCCATGCC-3’) and Irgb10 (5’-GGCATGGACGAGCTGTACAAGATGGGTCAGTCTTCTTCTAAAC-3’) and (5’-GCGGATCCGATTTAAATTCGTTACTCAGAGTCCACACTGTC-3’)and the vector backbone pCDH-CMV-MCS-EF1a-GFP-Neo (modified from CD516B-2, System Biosciences) was digested by EcoRI and treated with phosphatase to prevent re-ligation. To generate lentiviral particles, plasmids psPAX2 (12260, Addgene), pMD2.G (12259, Addgene), and the mCherry-IRGBIO containing transfer plasmid were co-transfected into 293T cells. Viral particle-containing supernatant plus polybrene (6 μg/mL) was used to transduce Irgb10^−/− primary mouse fibroblasts. Transduced cells were selected with G418 (400 μg/mL, A1720, Sigma-Aldrich) to generate fibroblasts expressing mCherry-tagged IRGB10.