DNA constructs and lentiviral production

GCaMP5g and GCaMP6s constructs have been described^4,5. For the CMV-GCaMP5g and CMV-GCaMP6s construct, the open reading frame of CMV-GCaMP5g (pCMV-GCaMP5g was a gift from Douglas Kim and Loren Logger, plasmid 31788, Addgene, Cambridge, MA, USA)⁴ or CMV-GCaMP6s (pGP-CMV-GCaMP6s was a gift from Douglas Kim, plasmid 40753, Addgene)⁵ used to replace the PGK-EGFP sequence in the parental pRRLsin.cPPT.PGK-EGFP.WPRE lentiviral vector plasmid (plasmid 12252, Addgene). For the CMV-mKO-GCaMP6s construct, the humanized monomeric Kusabira-Orange 2 cDNA sequence was obtained from phmKO2-MC1 (mKO, AM-V0145; MBL, Tokyo, Japan), linked via T2A to the open reading frame of GCaMP6s (plasmid 40753, Addgene) and then used to replace the EGFP sequence in the lentiviral backbone vector. For the hSyn-mKO-GCaMP6s construct, the human synapsin I promoter sequence obtained from pDRIVE-hSynapsin (Invivogen, San Diego, CA, USA) was attached to the mKO-T2A-GCaMP6s sequence and subcloned into the lentiviral backbone vector. For the hSyn-GCaMP5g and hSyn-GCaMP6s construct, the open reading frame of GCaMP5g or GCaMP6s was replaced the mKO-GCaMP6s in the hSyn-mKO-GCaMP6s lentiviral vector plasmid.

Lentiviruses were produced through transient transfection of 293T cells with CMV-GCaMP5g or CMV-mKO-GCaMP6s plasmid, along with gag-pol, rev-tat, and VSV-G packaging plasmids⁷¹. Viruses were harvested daily for 4 days, filtered (0.22 μm; EMD Millipore, Darmstadt, Germany) and concentrated by ultracentrifugation at 18000g for 4 hours at 4 °C, then reconstituted in media. For hSyn-mKO-GCaMP6s, hSyn-GCaMP5g, CMV-GCaMP6s and hSyn-GCaMP6s, ready to use high-titer purified lentiviral vectors were produced by the University of Pennsylvania School of Medicine Vector Core.