Plenti6 v5 dest gateway vector

The coding sequence of human COQ6 isoform a and isoform c, the mutated sequence COQ6 G255R, and a negative control expression sequence were transferred into the lentiviral expression plasmid (pLenti6/V5-DEST™ Gateway™ Vector, Invitrogen). To generate lentiviral particles, vectors were cotransfected with packaging vectors (ViraPower Packaging Mix, Invitrogen) into HEK293-FT cells using Lipofectamine 2000 (Life Technologies). Culture supernatants were harvested on day 3 and used to transduce HEK293 COQ6KO cells. Selection was carried out as described [15 (link)].

Production of lentiviral particles was carried out according to the manufacturer's protocol (Addgene Inc., Cambridge, USA) by usage of the packaging plasmids pMD2.G and psPAX2 (Invitrogen) and the lentiviral vector pLenti6/V5-DEST Gateway vector (Invitrogen, Paisley, UK), containing pHyPer-dMito (Evrogen, Moscow, Russia) and control expression sequence, respectively. For lentiviral infection, HDF were cultivated in 6-well plates. Upon reaching ∼70% confluence, culture medium, containing lentiviral particles to the amount of 2 MOI, was added to the cells in presence of 8 μg mL⁻¹ hexadimethrine bromide, which increases the efficiency of viral infection. Twenty-four hours after infection, medium was changed. Blasticidin selection was initiated (10 μg mL⁻¹) 48 h postinfection. For the detection of mitochondrial H₂O₂ levels, the cells were transfected with control or pHyPer-dMito/pLenti6/V5-DEST Gateway lentiviral vector and after expansion analyzed using confocal microscopy or flow cytometry (FACS Canto II, Becton Dickinson, Franklin Lakes, USA). The level of mitochondrial H₂O₂ was estimated as a mean value of green pHyPer-dMito fluorescence in 10⁴ cells.

For the generation of pLenti SPOP expression vectors, mutant or SPOP WT was TOPO TA-cloned from pCMV-SPOP vectors (Barbieri et al., 2012 (link)) into pLenti viral vectors (pLenti6/V5-Dest Gateway Vector, V496-10, Invitrogen). For SPOP mRNA, in vitro transcription SPOP cDNA was TOPO TA cloned into a pCR 8 TOPO TA Gateway entry vector by using the pCR 8/GW/TOPO TA cloning kit (K2500-20, Invitrogen). 3-way-gateway recombination was then performed by using LR clonase II (11791, Invitrogen) to recombine SPOP cDNA from the pCR 8 TOPO TA gateway middle entry vector into a pDestTol2pA2 attR4-R3 (#394) destination vector. The 5′ entry clone was (p5E-CMV/SP6, #382) and the 3′ entry clone was (p3E-poly A, #302). The final vector contains a SP6 site for subsequent in vitro mRNA transcription.