Your browser doesn't support javascript.
loading
Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.
Coleman, John W; Wright, Kevin J; Wallace, Olivia L; Sharma, Palka; Arendt, Heather; Martinez, Jennifer; DeStefano, Joanne; Zamb, Timothy P; Zhang, Xinsheng; Parks, Christopher L.
Afiliação
  • Coleman JW; The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States. Electronic address: jcoleman@IAVI.org.
  • Wright KJ; The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
  • Wallace OL; The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
  • Sharma P; The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
  • Arendt H; The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
  • Martinez J; The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
  • DeStefano J; The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
  • Zamb TP; The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States.
  • Zhang X; The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States; Program in Molecular and Cellular Biology, School of Graduate Studies, The State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States.
  • Parks CL; The International AIDS Vaccine Initiative, The AIDS Vaccine Design & Development Laboratory, Brooklyn, NY 11220, United States; Program in Molecular and Cellular Biology, School of Graduate Studies, The State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States.
J Virol Methods ; 213: 26-37, 2015 Mar.
Article em En | MEDLINE | ID: mdl-25486083
ABSTRACT
Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert.
Assuntos
Palavras-chave

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Replicação Viral / Portadores de Fármacos / Expressão Gênica / Produtos do Gene gag / Instabilidade Genômica / Vírus da Cinomose Canina / Vetores Genéticos Limite: Animals Idioma: En Ano de publicação: 2015 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Replicação Viral / Portadores de Fármacos / Expressão Gênica / Produtos do Gene gag / Instabilidade Genômica / Vírus da Cinomose Canina / Vetores Genéticos Limite: Animals Idioma: En Ano de publicação: 2015 Tipo de documento: Article