RESUMO
The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.
Assuntos
Sítios de Ligação Microbiológicos/genética , Bacteriófagos/enzimologia , Integrases/metabolismo , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Linhagem Celular , DNA Viral/genética , Resistência Microbiana a Medicamentos/genética , Genoma Humano , Humanos , Integrases/genética , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Neomicina/farmacologia , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética/genética , Transfecção , Integração ViralRESUMO
We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.
Assuntos
Bacteriófagos/enzimologia , Bacteriófagos/genética , Integrases/fisiologia , Integração Viral/genética , Integração Viral/fisiologia , Células 3T3 , Animais , Sequência de Bases , Linhagem Celular , DNA/genética , Primers do DNA/genética , Expressão Gênica , Genes Reporter , Genoma , Humanos , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Recombinação Genética , Seleção Genética , Homologia de Sequência do Ácido NucleicoRESUMO
The integrase from the Streptomyces phage phiC31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the presence of the phiC31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein-Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.