The adenovirus expressing 4mtGCaMP6f was generated using the
AdEasy strategy (Agilent). 4mtGCaMP6f (Tosatto et al., 2016 (
link)) was subcloned in the
pShuttle-CMV vector (Agilent) from pcDNA3.1-4mtGCaMP (Tosatto et al., 2016 (
link)) using the following primers:
fw: 5′- AATTTAGATCTCCAAGCTGGCTAGCATGTCC-3′
rv: 5′- AAATTGCGGCCGCTCACTTCGCTGTCATCATTT -3′
The PCR fragment was cloned into BglII and NotI sites in pShuttle-CMV (pShuttle-CMV was a gift from Bert Vogelstein (Addgene plasmid # 16403)). Subsequent steps were performed according to the manufacturer’s instructions (Agilent).
De Mario A., Tosatto A., Hill J.M., Kriston-Vizi J., Ketteler R., Vecellio Reane D., Cortopassi G., Szabadkai G., Rizzuto R, & Mammucari C. (2021). Identification and functional validation of FDA-approved positive and negative modulators of the mitochondrial calcium uniporter. Cell Reports, 35(12), 109275.
