Construction and Packaging of SyGCaMP6f Lentivirus

The synaptophysin::GCaMP6f (SyGCaMP6f) construct was provided by Dr. Leon Lagnado as an improved version of syGCaMP2^{52 (link),53 (link)}. The coding sequence of SyGCaMP6f was cloned into pWPXL (Addgene plasmid # 12257; http://n2t.net/addgene:12257; RRID:Addgene_12257) by replacing EGFP using BamHI and NdeI. For lentivirus production, HEK 293T cells were transfected by the calcium phosphate technique following methods described by Didier Trono (http://tronolab.epfl.ch/lentivectors) with pMD2G (Addgene plasmid # 12259; http://n2t.net/addgene:12259; RRID:Addgene_12259), pCMVR8.74 (Addgene plasmid # 22036; http://n2t.net/addgene:22036; RRID:Addgene_22036), and pWPXL. Two days later, culture medium containing lentiviral particles was collected in three rounds at 8 h intervals, kept at 4 °C, and centrifuged at 500×g. Supernatants were distributed in aliquots and stored at −80 °C.

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Velasco C.D, & Llobet A. (2020). Synapse elimination activates a coordinated homeostatic presynaptic response in an autaptic circuit. Communications Biology, 3, 260.

Publication 2020

pWPXL pMD2G pCMVR8.74

293t cells Calcium phosphate Coding sequence Culture medium Lentivirus Plasmid Synaptophysin

Corresponding Organization :

Other organizations : Universitat de Barcelona

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Variable analysis

independent variables

SyGCaMP6f construct

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive control: pMD2G (Addgene plasmid # 12259)
Positive control: pCMVR8.74 (Addgene plasmid # 22036)
Negative control: Not mentioned

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