A simplified system for generating recombinant adenoviruses is described. This system simplifies the generation and production of recombinant adenoviruses by using homologous recombination in bacteria rather than in eukaryotic cells. A recombinant adenoviral plasmid is generated with minimal enzymatic manipulations. After transfection of such plasmids into a mammalian packaging cell line, viral production is followed using green fluorescent protein (GFP) encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained without plaque purification. This system expedites the generation and testing of recombinant adenoviruses for various purposes. Recombinant adenoviruses are used for gene transfer in vitro, vaccination in vivo, and gene therapy. Adenoviruses are preferred for these applications due to their ability to transfer genes to a broad spectrum of cell types and their high titers and transgene expression levels. The adenoviral genome consists of a linear, 36-kb, double-stranded DNA molecule. Viral transcription units are divided into early (E1, E2, E3, and E4) and late based on their temporal expression relative to the onset of viral DNA replication. Traditional methods for generating recombinant adenoviruses involve direct ligation of DNA fragments or homologous recombination in mammalian cells. However, these methods are technically challenging and time-consuming. The new system uses homologous recombination in E. coli, which is more efficient and allows for the inclusion of up to 10 kb of transgene sequences. The system also allows for multiple transgenes to be produced from the same virus. The inclusion of a GFP gene in the viral backbone allows for direct observation of transfection and infection efficiency. The system involves the use of adenoviral plasmids and shuttle vectors. The adenoviral plasmids contain most of the adenoviral genome, while the shuttle vectors allow for the insertion of exogenous transgenes. The system allows for the production of homogeneous viruses without plaque purification, significantly reducing the time required for viral production. The system is efficient and practical, making it suitable for a variety of applications. The system has been used to generate more than 20 different adenoviruses with inserts ranging up to 10 kb. The system is efficient enough to allow for the generation of small libraries of transgenes produced in adenoviruses. The system is also useful for isolating expressing cells through fluorescence-activated cell sorting, facilitating various types of experiments. The system is supported by grants from the National Institutes of Health and is used by researchers worldwide.A simplified system for generating recombinant adenoviruses is described. This system simplifies the generation and production of recombinant adenoviruses by using homologous recombination in bacteria rather than in eukaryotic cells. A recombinant adenoviral plasmid is generated with minimal enzymatic manipulations. After transfection of such plasmids into a mammalian packaging cell line, viral production is followed using green fluorescent protein (GFP) encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained without plaque purification. This system expedites the generation and testing of recombinant adenoviruses for various purposes. Recombinant adenoviruses are used for gene transfer in vitro, vaccination in vivo, and gene therapy. Adenoviruses are preferred for these applications due to their ability to transfer genes to a broad spectrum of cell types and their high titers and transgene expression levels. The adenoviral genome consists of a linear, 36-kb, double-stranded DNA molecule. Viral transcription units are divided into early (E1, E2, E3, and E4) and late based on their temporal expression relative to the onset of viral DNA replication. Traditional methods for generating recombinant adenoviruses involve direct ligation of DNA fragments or homologous recombination in mammalian cells. However, these methods are technically challenging and time-consuming. The new system uses homologous recombination in E. coli, which is more efficient and allows for the inclusion of up to 10 kb of transgene sequences. The system also allows for multiple transgenes to be produced from the same virus. The inclusion of a GFP gene in the viral backbone allows for direct observation of transfection and infection efficiency. The system involves the use of adenoviral plasmids and shuttle vectors. The adenoviral plasmids contain most of the adenoviral genome, while the shuttle vectors allow for the insertion of exogenous transgenes. The system allows for the production of homogeneous viruses without plaque purification, significantly reducing the time required for viral production. The system is efficient and practical, making it suitable for a variety of applications. The system has been used to generate more than 20 different adenoviruses with inserts ranging up to 10 kb. The system is efficient enough to allow for the generation of small libraries of transgenes produced in adenoviruses. The system is also useful for isolating expressing cells through fluorescence-activated cell sorting, facilitating various types of experiments. The system is supported by grants from the National Institutes of Health and is used by researchers worldwide.