Padtrack cmv vector

NOXA-expressing adenovirus (Ad-NOXA) vector was constructed by inserting Flag-tagged NOXA cDNA (Ref) into pAdTrack-CMV vector (Addgene, Cambridge, MA), while the control adenovirus (Ad-Con) contained the vector alone. Adenoviruses were produced at the VCU shared resource core. Lentiviral short-hairpin RNA (shRNA) constructs were purchased from Sigma-Aldrich (St. Louis, MO). Constructs were transfected with psPAX2 and pMD2.G plasmids (Addgene, Cambridge, MA) into 293T cells with EndoFectin Lenti (GeneCopoeia, Rockville, MD), and the supernatants containing lentivirus were collected. The cell line of interest was infected with the lentiviruses and stable cell lines were established by puromycin (2 μg/ml) selection.

All cDNA was produced according to standard methods and verified by sequencing. Most of the ASXL1 constructs have been described previously^{5 (link), 6 (link)}. The desired AKT1 variants were created by PCR amplification using AKT1 cDNA (Sino Biological, North Wales, PA) and subcloned into suitable vectors (Flag (2×)-tagged and Myc-tagged pcDNA3 vectors) for overexpression in mammalian cells. For GST-fused and His-tagged proteins, the pGEX4T-1 (GE Healthcare, Piscataway, NJ) and pET15b (Novagen, Madison, WI) vectors were used. The p16Ink4a promoter-driven luciferase reporter was created by inserting the p16Ink4a promoter (3,000 bp), amplified by PCR using genomic DNA of mouse tails, into the pGL2 basic vector (Promega, Madison, WI). Details of plasmid constructs are available upon request. For the construction of adenoviruses expressing mAsxl1, the 2xFlag-mAsxl1 region was amplified by PCR and subcloned into the pAdTrack-CMV vector (Addgene, Cambridge, MA) and recombined with the pAdEasy-1 vector (Addgene) in E. coli BJ5183. The recombinant plasmid was used for rescuing recombinant adenovirus by transfection of QBI-293A cells (MP Biomedicals, Santa Ana, CA).