An Improved Technique for Obtaining Enhanced Infectivity with Herpes Simplex Virus Type 1 DNA

Abstract

SUMMARYCells infected with herpes simplex virus type I (HSV-I) DNA by the calcium phosphate precipitation technique produce virus which leads to the formation of plaques (Graham, Veldhuisen & Wilkie, 1973). In the study reported here we show that treatment of cell monolayers with dimethyl sulphoxide (DMSO) solutions after infection with DNA-calcium phosphate complexes leads to a considerable increase in the number of plaques obtained. The conditions for this enhancement of infectivity have been optimized for baby hamster kidney (BHK) cells, and increases in plaque numbers of over Ioo-fold have been obtained. The treatment appears to increase the proportion of cells which respond to DNA infection by initiating plaque formation, and results in a large increase in the measured specific infectivity of HSV-I DNA. DMSO causes similar (but quantitatively different) responses in various other cell lines infected with HSV-I DNA. BHK cells infected with either virus particles, or virus DNA by the DEAE-dextran technique (Laithier & Sheldrick, I975), do not exhibit this massive enhancement following exposure to DMSO.

High efficiency gene transfer to the central nervous system of rodents and primates using herpes virus vectors lacking functional ICP27 and ICP34.5

“…The ICP27 complementing cell line B130/2 was made by cotransfecting the plasmid pSG130 B/S 35 with the plasmid pM5GNeo encoding a neomycin resistance gene, into BHK cells using the calcium phosphate method. 36 Drug-resistant cells were selected with 500 g/ml G418 (Geneticin sulfate; Gibco).…”

Restoration of Wild-type Pathogenicity to an Attenuated DNA Polymerase Mutant of Herpes Simplex Virus Type 1

“…2). The recombinant plasmid was mixed with RSC-26 genomic DNA and co-transfected into baby hamster kidney cells (BHK-21) by the calcium phosphate precipitation method (Graham & Van der Eb, 1973;Stow & Wilkie, 1976). In order to enrich for recombinants expressing wt DNA polymerase the yield from the co-transfection experiment was passaged once in the presence of 0.3 pM-aphidicolin.…”

Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12.

“…Restriction endonuclease treated DNAs were heated for 3 min at 650C prior to transfection in order to eliminate possible end-to-end reassociation. Confluent TK-L cells in 25-cm2 Corning flasks were transfected as described (15) with modifications (16). Cultures were switched to HAT selective medium 24 hr later.…”