Lentiviral Transduction for Calretinin Manipulation

For CR overexpression, pLV-CALB2 was used [23 (link)]; and for CR downregulation CALB2 shRNAs [7 (link)]. pLKO.1-shGFP (plasmid #30323) was obtained from Addgene. Lentivirus particles were produced as described before [7 (link)]. Briefly, HEK293T cells were co-transfected by the CaPO₄ method with 3 μg of the envelope plasmid pMD2.G-VSVG (Addgene plasmid #12259), 8 μg of the packaging plasmid psPAX2 (Addgene plasmid #12260) and 10 μg of the transfer plasmid. Lentivirus in the supernatant of HEK293T cells was harvested 48 h and 72 h after transfection. The supernatant was filtered (0.45 μm) and resuspended in DMEM containing 10% FBS and 1% PS solution. To stably express Renilla Luciferase in MSTO-211H cells the GFP cassette in pLVTHM was replaced with a cDNA coding for the Renilla luciferase pGL4.74[hRluc/TK] (Promega). Briefly, the plasmid was digested with HindIII, filled with Klenow enzyme, and then digested with XbaI. The fragment was inserted into the PmeI and SpeI sites of the backbone of pLVTHM to produce the final plasmid pLV-hRluc. Lentivirus were produced using the same envelope plasmid as above and the packaging plasmid pCMV-dR8.91 (kind gift from Prof. D. Trono, EPFL, Switzerland).