Molecular cloning of adeno-associated virus variant genomes and generation of infectious virus by recombination in mammalian cells.
Continued passage of the human parvovirus, adeno-associated virus (AAV), at high multiplicity of infection in human cells results in the accumulation of AAV particles containing variant genomes. We have analyzed the structure of individual variant AAV genomes by molecular cloning in the Escherichia coli plasmid, pBR328. Each of the AAV inserts in six individual recombinant plasmids contained a single internal deletion but in contrast to a previous model, the locations of the deletions were nonrandom. The molecular cloning protocol also generated recombinant plasmids containing the entire AAV2 DNA sequence which yielded infectious AAV particles when transfected into human 293 cells in the presence of helper adenovirus using a DEAE-transfection procedure. Infectious AAV genomes were also generated by recombination when cells were jointly transfected with a mixture of plasmids containing two different mutant AAV genomes. The efficiency of this recombination appear to be influenced by the degree of homology between the mutant AAV genomes.
D001483:Base Sequence; D002277:Carcinoma; D002460:Cell Line; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D000229:Dependovirus / Q000235:genetics*; D005814:Genes, Viral; D014644:Genetic Variation; D006801:Humans; D009062:Mouth Neoplasms; D010957:Plasmids; D011995:Recombination, Genetic
D004279:DNA, Viral; D004262:DNA Restriction Enzymes