title: New tools to convert bacterial artificial chromosomes to a self-excising design and their application to a herpes simplex virus type 1 infectious clone.
abstract: Infectious clones are fundamental tools for the study of many viruses, allowing for efficient mutagenesis and reproducible production of genetically-defined strains. For the large dsDNA genomes of the herpesviridae, bacterial artificial chromosomes have become the cloning vector of choice due to their capacity to house full-length herpesvirus genomes as single contiguous inserts. Furthermore, while maintained as plasmids in Escherichia coli, the clones can be mutated using robust prokaryotic recombination systems. An important consideration in the design of these clones is the means by which the vector backbone is removed from the virus genome upon delivery into mammalian cells. A common approach to vector excision is to encode loxP sites flanking the vector sequences and rely on Cre recombinase expression from a transformed cell line. Here we examine the efficiency of vector removal using this method, and describe a "self-excising" infectious clone of HSV-1 strain F that offers enhancements in virus production and utility.
  
  
  
  Insertion of a fluorescent protein expression cassette into the vector backbone of the HSV-1 strain F clone, pYEbac102, demonstrated that 2 serial passages on cells expressing Cre recombinase was required to achieve > 95 % vector removal from the virus population, with 3 serial passages resulting in undetectable vector retention. This requirement was eliminated by replacing the reporter coding sequence with the CREin gene, which consists of a Cre coding sequence disrupted by a synthetic intron. This self-excising variant of the infectious clone produced virus that propagated with wild-type kinetics in culture and lacked vector attenuation in a mouse neurovirulence model.
  
  
  
  Conversion of a herpesvirus infectious clone into a self-excising variant enables rapid production of viruses lacking bacterial vector sequences, and removes the requirement to initially propagate viruses in cells that express Cre recombinase. The self-excising bacterial artificial chromosome described here allows for efficient production of the F strain of herpes simplex virus type 1.
authors: Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, 303 E. Chicago Ave., Morton Building, Room 3-603, Chicago, IL, 60611, USA.
authors: Alexsia L
authors: AL
authors: Richards
authors: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska, Lincoln, NE, 68583, USA.
authors: Patricia J
authors: PJ
authors: Sollars
authors: Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, 303 E. Chicago Ave., Morton Building, Room 3-603, Chicago, IL, 60611, USA. g-smith3@northwestern.edu.
authors: Gregory A
authors: GA
authors: Smith
mesh_terms: D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D019976:Cloning, Organism / Q000379:methods*; D024861:Genetic Enhancement / Q000379:methods*; D015497:HIV-1 / Q000235:genetics*; D019426:Integrases / Q000235:genetics; D051379:Mice; D011995:Recombination, Genetic / Q000235:genetics; D019562:Viral Load / Q000235:genetics*; D014774:Virulence / Q000235:genetics
chemical_list: C045073:Cre recombinase; D019426:Integrases
keywords: BAC; Bacterial artificial chromosome; HSV-1; Herpes simplex virus type 1; Herpesvirus; Infectious clone; Self-excising; Self-recombining