Codon-optimized hACE2 sequences were cloned into the shuttle vector (pShuttle-CMV, Addgene 240007) to generate pShuttle-hACE2. pShuttle-hACE2 was linearized with PmeI and subsequently cotransformed with the HuAdv5 backbone plasmid (pAdEasy-1 vector; Addgene 240005) into E. coli strain BJ5183 to generate pAdV5-ACE2 by homologous recombination as described (He et al., 1998 (link)). The pAdEasy-1 plasmid containing the HuAdV5 genome has deletions in E1 and E3 genes. hACE2 is under transcriptional control of a cytomegalovirus promotor and is flanked at its 3′ end by a SV40 polyadenylation signal. The pAd-hACE2 was linearized with PacI restriction enzyme before transfection into T-Rex 293 HEK cells (Invitrogen) to generate HuAdv5-hACE2. Recombinant HuAdv5-hACE2 was produced in 293-HEK cells and purified by CsCl density-gradient ultracentrifugation. The viral titer was determined by plaque assay in 293-HEK cells.
Padeasy 1 vector
Manufactured by Addgene
The PAdEasy-1 vector is a plasmid that is commonly used in molecular biology research. It is designed for the generation of recombinant adenoviruses, which can be used to express genes of interest in a variety of cell types. The vector contains the necessary elements for the production and packaging of adenoviral particles, including the adenoviral inverted terminal repeats (ITRs) and the adenoviral packaging signal.
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2 protocols using padeasy 1 vector
1
Generation of Recombinant Adenovirus Expressing hACE2
2
Overexpression of AKT1 and ASXL1 in Mammalian Cells
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All cDNA was produced according to standard methods and verified by sequencing. Most of the ASXL1 constructs have been described previously5 (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).
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