Optimizing Viral Transduction of hOPCs

EF1α:GCaMP6s was described previously^{12 (link)}. EF1α:jRCaMP1a was cloned as follows. pGP-CMV-NES-jRCaMP1a was obtained from AddGene (#61562)^{35 (link)}. The nuclear export signal (NES) and jRCaMP1a coding region was PCR amplified: SpeI, forward-5′ AAAACTAGTGAACCGTCAGATCCGCTAG-3′; PspXI (Thermo Fisher Scientific), reverse, 5′-AAAGCTCGAGCTCTACAAATGTGGTATGGCTG-3′ (Thermo Fisher Scientific). PCR products were purified (Monarch PCR Cleanup Kit (NEB, #T1030S)) and restriction digested with unique 5′ SpeI and 3′ PspXI sites, and purified again. NES-jRCaMP1a digested PCR fragments were then ligated into lentiviral pTRIP-EF1α vector (a gift of Abdel Benraiss, University of Rochester)^{36 (link)}. The plasmid pLenti-EF1α-OptoSTIM was a gift from Taeyoon Kyung (KAIST, DAEJEON, Republic of Korea)^{37 (link)}. Lentiviruses were generated as described^{38 (link)} and titration of EF1α:GCaMP6s and EF1α:OptoSTIM was performed as previously described^{39 (link)}. Optimized multiplicity of infection (MOI) in hOPCs was determined for EF1α:GCaMP6s and EF1α:jRCaMP1a by infecting hOPCs and quantifying proportion of responsive cells following administration of 25 µM Oxo-M. Optimal MOI for EF1α:OptoSTIM lentivirus was determined through analysis of optimal GFP expression in infected hOPCs.