Patient cells harboring the E35* or the G57R mutation in SDHAF1 were engineered to stably express C-terminally FLAG/MYC-tagged human SDHAF1 or SDHAF1 without any tag at the C-terminus, by lentiviral mediated transduction with the pLenti-C-MYC-DDK-IRES-Neo (Origene), or with pLenti-IRES-Neo (from which the tags had been removed by site-directed mutagenesis), encoding wild type SDHAF1, using the lenti-vpak packaging system (Origene) (See Table S1 for a complete list of plasmids used in this study). Neomycin resistant clones were isolated, analyzed for SDHAF1 expression by western blot, and tested for CII enzymatic activity. A stable clone with restored SDHAF1 (or SDHAF1-F/M) expression and CII activity was expanded for each patient cell line. In parallel, patient cells were transduced with the empty vector (pLenti-C-MYC-DDK-IRES-Neo or with the same plasmid without tags) and used as controls. Stable expression of FLAG-tagged SDHB was established in SDHAF1-deficient cells and in patient cells after re-expression of SDHAF1, by lentiviral mediated transduction with pLYS5-SDHB-FLAG (Addgene). Hygromycin resistant clones were isolated and analyzed for expression of FLAG-tagged SDHB by western blot. A stable clone for each of the cell lines of interest was expanded and used in further experiments.
Lenti vpak packaging system
Manufactured by OriGene
Sourced in United States
The Lenti-vpak packaging system is a lab equipment product designed for the production and purification of lentiviral particles. The core function of this system is to facilitate the efficient generation and collection of high-titer lentiviral stocks for various research applications.
Automatically generated - may contain errors
2 protocols using lenti vpak packaging system
1
Engineering SDHAF1-Deficient Cells to Restore Respiratory Complex II Activity
2
BCAT1 Mutant Generation for Functional Study
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BCAT1 cDNA transcript variant 5 (NM_001178094) contained in PCMV6-Entry vector (PS100001, Origene) was obtained from Origene (Rockville, MD, USA). Site-directed mutagenesis of the BCAT1-CXXC motif was performed using the Quikchange II Site-directed Mutagenesis Kit (Agilent, UK) and mutagenic primers (C338SF 5′-GGTACAGCCTGTGTTGTTAGCCCAGTTTCTGATATACTG-3′, C338SR 5′-CAGTATATCAGAAACTGGGCTAACAACACAGGCTGTACC-3′). Successful mutation was confirmed by capillary sequencing at the University of Birmingham DNA Sequencing Facility. BCAT1-wild type (WT) and CXXC motif mutant BCAT1-C338S were sub-cloned into a pLenti-C-Myc-DDK-IRES-Puro lentiviral gene expression vector (Origene, UK, see Supplemental Figure S1 ) and Lentiviral particles containing pLenti-BCAT1 vectors were generated using Lenti-vpak packaging system (Origene, MD, USA) in HEK293T cells. For lentiviral transduction 2 × 105 cells were added to 1 mL of viral suspension and centrifuged at 900× g for 2 h at 32 °C. Stably transduced cells overexpressing BCAT1 constructs were selected for incubation with complete RPMI supplemented with 0.5 µg/mL Puromycin.
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