Journal:

Article Title: Feasibility of Generating Adeno-Associated Virus Packaging Cell Lines Containing Inducible Adenovirus Helper Genes

doi: 10.1128/JVI.76.4.1904-1913.2002

Figure Lengend Snippet: Construction of inducible Ad gene expression plasmids and AAV plasmids. The Ad type 2 E1A gene (without a promoter) and the E1B gene (with the endogenous promoter) were PCR amplified and cloned into the EcoRV site of plasmid PBI (Clontech), which has a TET-inducible promoter consisting of a minimal CMV promoter and a TRE enhancer. Plasmid pST-E1AB contains the E1A and -B genes in the sense orientation with respect to the TET-inducible promoter, whereas plasmid pST-E1A/B-R has the E1A and -B genes in the reverse orientation. Plasmid pXX2-GFP-Hyg harbors an AAV vector with a GFP reporter gene and a hygromycin resistance gene flanked by two inverted terminal repeats (ITR) of AAV. This plasmid also harbors the AAV viral coding genes Rep and Cap at a different locus. Plasmid pXX6-ZEO contains a zeomycin resistance gene and three Ad genes (VA RNA, E2A, and E4) that can provide helper functions to AAV.

Article Snippet: The primary antibody for AAV capsid proteins was a guinea pig polyclonal antibody against AAV type 2 (Braton Biotech, Inc.); it was used at a dilution of 1:400.

Techniques: Expressing, Amplification, Clone Assay, Plasmid Preparation