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Amplicons are defective, helper-dependent, herpes simplex virus type 1 (HSV-1)-derived vectors. The main interest of these vectors as gene transfer tools stems from the fact that the amplicon vector genomes do not carry protein-encoding viral sequences. Consequently, they are completely safe for the host and nontoxic for the infected cells. Moreover, the complete absence of virus genes provides space to accommodate very large foreign DNA sequences, up to almost 150-kb, the size of the virus genome. This large transgene capacity can be used to deliver complete gene loci, including introns and exons, as well as long regulatory sequences, conferring tissue-specific expression or stable maintenance of the transgene in proliferating cells. During many years the development of these vectors and their application in gene transfer experiments was hindered by the presence of contaminating toxic helper virus particles in the vector stocks. In recent years, however, two different methodologies have been developed that allow generating amplicon stocks either completely free of helper particles or only faintly contaminated with fully defective helper particles. This chapter describes these two methodologies. |
