Lentivirus production

To express the Ca²⁺ sensor Fast‐GCaMP6f‐RS09 under control of the EF1a promoter together with a puromycin resistance, the lentiviral vector LeGO‐EF1a i‐Pur2 (derived from Addgene plasmid #27341 LeGO‐iG2) was digested with BamHI and NotI. PCR was performed using Phusion polymerase and plasmid Fast‐GCaMP6f‐RS09 (Addgene 67160) as a template encoding GCaMP6f with an N‐terminal 6× His Tag, a T7 Tag and an Xpress Tag with the following primer pair: 5′‐GTCGTGAGGAATTCGGATCCaccATGGGTTCTCATCATCATCATCATC and 5′‐ ATTTACGTAGCGGCCGCTTACTTCGCTGTCATCATTTGTAC. PCR product and digested plasmid LeGO‐EF1a i‐Pur2 were incubated with 5X In‐Fusion HD Enzyme Premix (In‐Fusion HD Cloning Kit, Clontech) according to the recommendations of the manufacturer. Resulting clones were checked by restriction digest, PCR and were finally verified by sequencing. To express human parvalbumin (Homo sapiens parvalbumin (PVALB), transcript variant 2, mRNA; NCBI Reference Sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_001315532.2","term_id":"1519312481","term_text":"NM_001315532.2"}}NM_001315532.2) under control of a ubiquitous EF1a promoter, a gene block was designed (Integrated DNA Technologies, IDT). This gene block comprised 5′‐overhangs of 15 bp each homologous to the lentiviral plasmid LeGO‐EF1a‐iPur2 (kind gift from Boris Fehse, University Medical Center Hamburg‐Eppendorf, Hamburg, Germany). After digestion of LeGO‐EF1a‐iPur2 with BamH I and Not I, both linearized plasmid and gene block were assembled using the In‐Fusion Cloning Kit (Takara Clontech), generating LeGO‐EF1a‐huPVALB‐iPur2. Resulting expression clones were checked by restriction digest, PCR and were finally verified by sequencing. Stocks of VSV‐G pseudotyped viral particles were produced at the Vector Facility of the University Medical Center Eppendorf using lentiviral transfer plasmid LeGO‐EF1a‐huPVALB‐iPur2 and packaging plasmids psPAX2 (Addgene plasmid #12260) and pMD2.G (Addgene plasmid #12259). LeGO‐EF1a‐iPur2 was employed to produce negative control lentivirus. After concentration by ultracentrifugation for 2 h at 4°C (140,000 g, SW32Ti rotor) on a 20% sucrose cushion, the pellet was resuspended in DPBS. The functional titer was determined by transduction of HEK293T and quantification by flow cytometry (FACSCantoII, BD Biosciences; FITC Channel).