Expression and rescue of a nonselected marker from an integrated AAV vector

Abstract

We used rep + and rep − recombinant AAV-plasmid vectors containing the nonselectable marker chloramphenicol acetyltransferase (CAT) driven by the AAV p 40 promoter, and having a selectable marker, neo, inserted in the plasmid genome, and driven by a herpesvirus thymidine kinase gene promoter. Each vector was transfected into human 293 cells or HeLa cells and the neo gene was used to select geneticin-resistant ( gen r) cells containing integrated vectors. The gen r cells were then screened for expression of the unselected marker CAT. For 293 cells, most clones from the rep − vector gave high CAT expression whereas only 50% of those from the rep + vector expressed CAT, generally at low level. For HeLa cells about 25% of the clones derived from either the rep + or rep − vector expressed CAT, and several clones from the rep + vector gave very high yields. We also analyzed integrated rep − vectors by rescue after superinfection with adenovirus and by Southern blotting. The AAWCAT genome could be rescued from 50% of HeLa cell clones but not from 293 cell clones. Lack of rescuability reflected rearrangement of the AAV genome termini or the rep gene. Western blotting showed low level constitutive expression of rep protein in one 293 cell clone and two HeLa cell clones. Thus, the AAV p 40 promoter (as well as p 5 and pig) can function in integrated vectors to express unselected markers which can subsequently be rescued. Expression and rescue depended upon several parameters including the cell type, the initial structure of the vector ( rep + or rep −) but not continued expression of rep, and possibly global effects of the surrounding chromatin.