RESUMEN
We have recently developed a high-efficiency method of constructing adenovirus vectors based on Cre-mediated recombination between two plasmids co-transfected into 293 cells. The simplicity and efficiency of this method should greatly expedite the construction of most recombinant vectors. However, this system would not be suitable for constructing vectors bearing loxP sites elsewhere in the genome because of undesirable Cre-mediated vector rearrangements. To address this, we have developed a similar system using FLP-mediated site-specific recombination for the construction of adenovirus vectors.
Asunto(s)
Adenoviridae/genética , ADN Nucleotidiltransferasas/metabolismo , Vectores Genéticos , Saccharomyces cerevisiae/enzimología , Proteínas Virales , Línea Celular , ADN/genética , Humanos , Integrasas/metabolismo , Plásmidos/genética , Transfección , beta-Galactosidasa/genéticaRESUMEN
The two-plasmid rescue method of constructing Ad vectors, which relies on either homologous or Cre-mediated recombination between two plasmids cotransfected into 293 or 293Cre4 cells, respectively, offers advantages over other approaches because of its simplicity. We have improved the efficiency of vector construction by both homologous and Cre-mediated recombination by replacing the single ITR in the shuttle plasmid with a head-to-head ITR junction. We have also expanded the versatility of this method by incorporating a Cre expression cassette into the plasmids to permit high-efficiency Cre-mediated vector rescue using 293 cells, abrogating the need for Cre-expressing cell lines. This new system retains the simplicity of the original but results in an approximately 100-fold increase in the number of recombinant viruses produced, all of which contain the foreign DNA insert, and allows high-efficiency Cre-mediated vector isolation using any E1-complementing cell line.
Asunto(s)
Adenoviridae/genética , Ingeniería Genética/métodos , Vectores Genéticos/genética , Plásmidos/genética , Proteínas Virales , Línea Celular/virología , Humanos , Integrasas/genética , Recombinación Genética , Secuencias Repetidas TerminalesRESUMEN
Adenovirus (Ad) vectors provide a highly efficient means of mammalian gene transfer and are widely used for high-level protein expression in mammalian cells, as recombinant vaccines and for gene therapy. A commonly used method for constructing Ad vectors relies on in vivo homologous recombination between two Ad DNA-containing bacterial plasmids cotransfected into 293 cells. While the utility of this two-plasmid approach is well established, its efficiency is low owing to the inefficiency of homologous recombination. To address this, we have developed an improved method for Ad vector construction based on Cre-mediated site-specific recombination between two bacterial plasmids, each bearing a loxP site. Ad vectors are generated as a result of Cre-mediated site-specific recombination between the two plasmids after their cotransfection into 293 cells expressing Cre recombinase. The frequency of Ad vector rescue by Cre-mediated site-specific recombination is significantly higher (approximately 30-fold) than by in vivo homologous recombination. The efficiency and reliability of this method should greatly simplify and expedite the construction of recombinant Ad vectors for mammalian gene transfer. Ad vectors are commonly constructed by homologous recombination between two plasmids cotransfected into 293 cells. This method has numerous advantages but results in low numbers of plaques owing to inefficient recombination. We have developed an improved method based on Cre-mediated site-specific recombination, which results in vector rescue at frequencies approximately 30-fold higher than by homologous recombination. This method should greatly simplify and expedite the construction of recombinant Ad vectors for mammalian gene transfer.