Lenti x packaging single shots vsv g system

We utilized the sequence of the HIV-1 2574-nt antisense transcript reported previously (GenBank accession number JQ866626.1) (Kobayashi-Ishihara et al., 2012 (link)). The sequence was mutated at nucleotide 1526 (C > T), 1540 (C > A), and 1549 (C > A) to introduce three stops at codons 3, 7 and 10 in the open reading frame of the HIV-1 antisense protein. The mutated sequence of the ASP transcript (ASTmut) was cloned into the EcoRI and XbaI sites of the pLVX-Puro vector (Clontech). For the generation of lentiviral particles, the pLVX-ASTmut plasmid or the empty pLVX-Puro plasmid were transfected into Lenti-X 293T cells using the Lenti-X Packaging Single Shots (VSV-G) system (Clontech). After 3–4 days, culture supernatants were collected, and viral particles purified and concentrated using the Fast-Trap Lentivirus Purification and Concentration Kit (Millipore). Jurkat and Jurkat E4 cells were transduced with purified lentiviral particles overnight in complete RPMI medium containing 2 μg/ml polybrene. Cells were then selected in fresh medium containing puromycin. Stably transduced cells were maintained in medium containing 1 μg/ml puromycin.