A cell-free recombination system for site-specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome.

A cell-free recombination system for site-specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome.

A cell-free recombination system for site-specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome.
The a sequence of herpes simplex virus type 1 (HSV-1) is a region bracketed by two direct repeats named DR1. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). We report here a new HSV/AAV hybrid amplicon vector system that not only solved problems associated with Rep expression but also markedly improved the stable transduction efficiency of this vector. By analogy to well-characterized bacteriophage systems, the association of these proteins with various forms of capsids, including procapsids, might be expected to clarify their roles in the packaging process. It consists of two covalently linked segments designated long (L) and short (S). Horizontal arrows indicate the directions of primers, transgene, AAV genome, and ITR; bold nucleotides represent sequences from AAV ITR; italic nucleotides represent sequences from AAVS1; vertical arrows and either bold or italic nucleotide (nt) numbers indicate the breakpoints in AAV ITR or AAVS1, respectively; underlined nucleotides are common to AAV ITR and AAVS1; numbers on the left (1 to 8) are the clone numbers. The latter recombinant virus was also ts for growth.

The ability to express viral genes from plasmids which can be shuttled into and out of the HSV genome in cell-free recombination reactions makes this a powerful method for performing genetic studies of the biologic properties of viral gene products.

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