Cloning and integration of DNA fragments in human cells via the inverted terminal repeats of the adeno-associated virus 2 genome

Abstract

In current systems for molecular cloning of eukaryotic genes, bacterial cells are routinely utilized as intermediate hosts. We investigated the possibility of using a viral system for cloning DNA fragments independent of bacterial cell usage. In this report, we provide an alternative approach for molecular cloning of DNA fragments in eukaryotic cells by utilizing the inverted terminal repeats (ITRs) of the genome of a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). We constructed a series of chimeric linear duplex DNA molecules, ranging in length from 1.8 to 7.2 kb, containing the cruciform structures of AAV-ITRs at both ends. These ‘no-end’ ( NE) DNA structures, when transfected into adenovirusinfected human cells in the presence of AAV replication proteins (Rep), underwent DNA replication. Furthermore, in the presence of AAV capsid proteins (Cap), all replicated DNA molecules of less than 5.0 kb were packaged into mature, biologically active AAV progeny virions. When a chimeric NE DNA ( NE-neo) containing a gene ( neo) encoding resistance to neomycin was transfected into human cells, neo R clones could be readily isolated in the presence of G418 (Geneticin). Southern-blot analysis of genomic DNA of several independently isolated neo R clones suggested stable integration of the NE-neo DNA into the host chromosomal DNA. AAV-ITRs, therefore, offer an alternative system for molecular cloning, as well as packaging of DNA fragments in mammalian cells independent of bacterial cell usage.