title: Impact of E1 and Cre on adenovirus vector amplification: developing MDCK CAV-2-E1 and E1-Cre transcomplementing cell lines.
abstract: Adenovirus vectors have been extensively studied through the manipulation of viral genome. However, little attention is being paid to their producer cell-lines; cells are selected according to virus yields, neglecting the expression profile of transcomplementing gene products underlying cell performance. This work evaluates the impact of E1 (E1A and E1B) and Cre recombinase levels in the production of E1-deleted and helper-dependent canine adenovirus type 2 (CAV-2) vectors using MDCK cells. E1A and E1B gene expression and Cre activity were evaluated in different cell clones and compared with the corresponding cell productivity and susceptibility to oxidative stress injury. CAV-2 production was proportional to E1A expression (the highest levels of E1A corresponding to productivities of 3000-5000 I.P./cell), while E1B prolonged host cell viability after infection, conferring protection against apoptosis. Cre recombinase counteracted E1B anti-apoptotic properties, however viral production was maintained under high levels of Cre. Yet, Cre recombinase side effects can be reduced using cell lines with lower Cre-activities, without compromising the excision efficiency of helper vector packaging signal. These results highlight the influence of transcomplementing gene products on CAV-2 producer cell line performance, and the ability to express high levels of E1A and E1B as an important feature for cell line establishment and high adenovirus titers.
authors.affiliation: iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
authors.forename: Paulo
authors.initials: P
authors.lastname: Fernandes
authors.forename: Virgínia M
authors.initials: VM
authors.lastname: Santiago
authors.forename: Ana F
authors.initials: AF
authors.lastname: Rodrigues
authors.forename: Hélio
authors.initials: H
authors.lastname: Tomás
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Kremer
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
authors.forename: Ana S
authors.initials: AS
authors.lastname: Coroadinha
mesh_terms: D017498:Adenovirus E1 Proteins / Q000235:genetics* / Q000378:metabolism; D006516:Adenoviruses, Canine / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D004285:Dogs; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics*; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D018384:Oxidative Stress; D014779:Virus Replication / Q000235:genetics
chemical_list: D017498:Adenovirus E1 Proteins; C045073:Cre recombinase; D019426:Integrases
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