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Master's Dissertation
DOI
10.11606/D.42.2009.tde-09022010-110303
Document
Author
Full name
Patrícia Duarte
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2009
Supervisor
Committee
Strauss, Eugenia Costanzi (President)
Nunes, Fabio Daumas
Yan, Chao Yun Irene
Title in Portuguese
Desenvolvimento da linhagem celular LEY79SF para produção de adenovírus livre de partículas competentes de replicação
Keywords in Portuguese
Adenovírus (produção)
Adenovírus competentesa de replicação
Linhagem celular
Linhagem celular Y79
Livre de RCA
Vetor retroviral
Abstract in Portuguese
A presença de Ad com competência para replicar (RCA, replication-competent adenovirus) nas preparações é um dos maiores problemas para a produção de Ad em larga escala. RCAs são gerados pela recombinação entre seqüência do vetor e seqüência homóloga do gene E1 presente nas células helper. Objetivo: desenvolver uma nova linhagem auxiliar para produção de Ad livre de RCA - LEY79 - derivada da linhagem de retinoblastoma humano Y79, tratando-se da primeira linhagem empacotadora de adenovírus com inativação mutacional da proteína supressora de tumor pRb, que crescem em suspensão. Células Y79 foram infectadas com o retrovírus pCLDE1A/E1BSN, selecionadas com G418. A eficiência de produção de AdeGFP na linhagem LEY79 foi testada e comparada com a HEK293A. Células Y79 foram adaptadas em meio livre de soro. Esperamos com a linhagem LEY79SF inovar no campo de processos para a produção de Ad recombinante.
Title in English
Development LEY79SF line for production of RCA-free Ad
Keywords in English
Adenovirus (production)
Cell line
RCA - free
Replication - competent adenovirus
Retroviral vector
Y79 cell line
Abstract in English
The presence of Ad with the ability to replicate (RCA, replication-competent adenovirus) in preparations is a major problem in the large-scale production of Ad. RCAs are generated by recombination between the vector sequence and sequence of the homologous gene in E1 helper cells. Objective: To develop a new helper cell line for the production of RCA-free Ad., called LEY79, derived from the Y79 of human retinoblastoma line, the first line Packer adenovirus with mutational inactivation of the tumor suppressor protein pRb, which are adapted to grow in suspension. Y79 cells were infected with the retrovirus pCLDE1A/E1BSN, selected with G418. The efficiency of production of AdeGFP in the LEY79 was tested and compared with the HEK293A. Y79 cells were adapted to grow in serum-free medium. We hope that use of the the LEY79SF cell line will promote innovation in the processing and production of recombinant Ad.
 
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Publishing Date
2010-03-15
 
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