Constitutive and Inducible Lentiviral Expression

TAZ-CAMTA1, TAZ-CAMTA1^S3A, TAZ, and TAZ^S4A cDNAs were cloned into the NheI/BamHI cut sites of pCDH-CMV-MCS-EF1-Puro (CD510B-1) from Systems Biosciences for constitutive expression. TAZ-CAMTA1 was cloned into the NheI/BamHI cut sites of the doxycycline-inducible all-in-one lentivirus construct pCW57-MCS1-2A-MCS2 (Barger et al. 2019 (link)). Primers used for cloning are listed in Supplemental Table S1. For virus production, the appropriate plasmid and the helper plasmids (psPAX2 and pMD2.G) were transfected into 293T cells cultured at 50% confluence in each well of a six-well plate dish using FuGene HD (Promega) according to the manufacturer's instructions. After 24 h, media was replaced. Viral supernatants were collected 76 h after transfection and filtered through a 0.45-µm filter. Viral supernatants were added to MS1 cells with 10 µg/mL polybrene (Santa Cruz Biotechnology). Pooled stable cells were selected with 1 µg/mL puromycin (Thermo Fisher Scientific). psPAX2 (Addgene plasmid 12260) and pMD2.G (Addgene plasmid 12259) were gifts from Didier Trono. pCW57.MCS1-2A-MCS2 was a gift from Adam Karpf (Addgene plasmid 71782).