Gene delivery using helper virus-free HSV-1 amplicon vectors.
Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only 1% of the viral genome. Consequently, replication and packaging of amplicons depend on helper functions provided either by replication-defective mutants of HSV-1 (helper viruses) 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. 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. Resulting stocks of packaged amplicon vectors are free of contaminating helper virus. The basic protocol describes the cotransfection of amplicon and cosmid DNA into 2-2 cells and the harvesting of packaged vector particles. Support protocols describe preparing cosmid DNA and methods for determining the titers of amplicon stocks.
Fraefel
Cornel
C
University of Zurich, Institute of Virology, Zurich, Switzerland.
D000818:Animals; D002460:Cell Line; D003360:Cosmids; D004274:DNA, Recombinant / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005826:Genetics, Medical; D006378:Helper Viruses / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D014162:Transfection; D019065:Virus Assembly
D004274:DNA, Recombinant