Summary auto-generated
This study developed stable 293 cell lines expressing adeno-associated virus (AAV) large Rep proteins using the Cre/loxP switching system to overcome Rep toxicity. AAV is a small DNA virus used for gene therapy, but its Rep proteins inhibit cell growth, making constitutive expression problematic. The researchers designed plasmids where a "stuffer" sequence flanked by loxP sites blocked Rep expression until removed by Cre recombinase from adenoviral infection. Western blotting confirmed that Rep78/68 proteins were strongly induced upon Cre-expressing adenovirus infection in stable transfectants, while Cap proteins showed minimal induction. When supplemented with a plasmid expressing Rep52/40 and Cap proteins, the cells produced approximately 5.5×10⁸ AAV vector particles per dish—100-fold more than without supplementation. However, this remained lower than transient transfection controls. The results demonstrate that large Rep protein expression can be tightly regulated by the Cre/loxP system and that induced Rep proteins retain packaging functionality. The authors suggest that inducible expression systems for Cap proteins would also be necessary to develop highly efficient AAV-packaging cell lines, as Cap toxicity limited protein expression in stable clones.
Key findings
- The Cre/loxP system successfully regulated large Rep protein (Rep78/68) expression in stable 293 cells, with strong induction upon Cre-expressing adenovirus infection and no basal expression in uninduced cells
- Induced large Rep proteins retained full packaging functionality, demonstrating the system's utility for AAV vector production
- Supplementation with Rep52/40 and Cap expression plasmid increased vector production 100-fold to 5.5×10⁸ particles per dish, though lower than transient transfection controls
- Cap protein toxicity limited its expression in stable clones, suggesting that inducible expression systems for both Rep and Cap proteins are needed for optimal AAV-packaging cell lines
This summary was generated automatically from the article PDF and is not part of the original publication. Refer to the PDF for the authoritative text.
Abstract
Since the Rep proteins of adeno-associated virus (AAV) are harmful to cells, it is difficult to obtain stable cell lines that express them constitutively. In this study, stable 293 cell lines were obtained in which large Rep expression was inducible by using the Cre/loxP switching system. To determine the function of the induced Rep proteins, the packaging capacity was examined after supplementation with a plasmid expressing small Rep and Cap proteins. A significant amount of recombinant AAV (5·5x108 vector particles per 10 cm dish) was produced by transfection with a vector plasmid and infection with Cre-expressing recombinant adenovirus, indicating that the large Rep proteins retained the function required for packaging. These findings indicate that large Rep protein expression can be strictly regulated by the Cre/loxP system and will also serve as a basis for the development of an efficient AAV-packaging cell line.