Abstract
Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only a very small percentage of the 152-kbp viral genome. Consequently, replication and packaging of amplicons depend on helper functions that are provided either by replication-defective mutants of HSV-1 or by replication-competent, but packaging-defective, HSV-1 genomes. Sets of cosmids that overlap and represent the entire HSV-1 genome can form, via homologous recombination, circular replication-competent viral genomes, which give rise to infectious virus progeny. However, if the DNA cleavage/packaging signals are deleted, reconstituted virus genomes are not packageable, but still provide all the helper functions required for the packaging of cotransfected amplicon DNA. The resulting stocks of packaged amplicon vectors are essentially free of contaminating helper virus. This unit describes the cotransfection of amplicon and cosmid or bacterial artificial chromosome (BAC) DNA into 2-2 cells by cationic liposome-mediated transfection and the harvesting of packaged vector particles. Support protocols provide methods for preparing cosmid and BAC DNA and determining the titers of amplicon stocks.
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