title: An in vitro ligation and transfection system for inserting DNA sequences into the latency-associated transcripts (LATs) gene of herpes simplex virus type 1.
abstract: This report describes a simple, rapid and highly efficient method for introducing specific DNA sequences into a defined locus of the herpes simplex virus type 1 (HSV-1) genome by restriction enzyme cleavage and ligation. The genome of the HSV-1 strain HFEM contains a 4.1 kb deletion in one copy of the RL region, deleting one copy of the latency-associated transcript (LAT) gene. It does not contain any site for restriction enzyme PacI. Two unique PacI restriction enzyme sites flanking an HSV-1 ICP6 promoter-LacZ reporter gene cassette were engineered into the LAT region to generate a recombinant virus HFEM/ICP6-LacZ which produced blue plaques in the presence of X-gal. This viral vector allowed the insertion of foreign genes directly into the HSV-1 genome by restriction enzyme digestion and ligation. The system was tested by digesting the HFEM/ICP6-LacZ DNA with PacI and with SwaI (an endogenous unique restriction enzyme site upstream of the LAT promoter locus and inserting by in vitro ligation a LAT promoter-LacZ gene cassette into the HFEM/ICP6-LacZ genome. The new recombinant virus HFEM/LAT-LacZ was detected as white plaques in the presence of X-gal, since beta-galactosidase expression, when driven by the LAT promoter, is not detectable during viral replication in tissue culture. The high yield (approximately 100%) of the recombinant virus obtainable from this in vitro ligation and transfection procedure coupled with a blue-white or reversible white-blue plaque detection scheme makes this a powerful method for constructing HSV-1 vectors around the LAT promoter locus.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Wistar Institute, Philadelphia, PA 19104, USA.
authors.forename: Q S
authors.initials: QS
authors.lastname: Huang
authors.forename: S L
authors.initials: SL
authors.lastname: Deshmane
authors.forename: N W
authors.initials: NW
authors.lastname: Fraser
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics*; D005814:Genes, Viral; D005818:Genetic Engineering; D005821:Genetic Techniques; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D004274:DNA, Recombinant