Skip to main content

(NAS Colloquium) Genetic Engineering of Viruses and Viral Vectors (1996) / Chapter Skim
Currently Skimming:

Use of virion DNA as a cloning vector for the construction of mutant and recombinant herpesviruses
Pages 11389-11394

The Chapter Skim interface presents what we've algorithmically identified as the most significant single chunk of text within every page in the chapter.
Select key terms on the right to highlight them within pages of the chapter.


From page 11389...
... We also report direct cloning of a replaceable foreign gene expression cassette into HVS. The methods described Abbreviations: HVS, herpesvirus saimiri; STP, saimiri transforming protein; Tip, tyrosine kinase interacting protein; SEAP, secreted engineered alkaline phosphatase; GFP, green fluorescent protein; SN140, simian virus 40.
Read the entire page →
From page 11390...
... To demonstrate rescue of wild-type phenotype, DNA from virions of the STP-C488 deletion virus was used for cotransfection with linearized wild-type p488PX plasmid, followed by limiting dilution purification of SEAP negative virus with STP-C488 restored. By OMK cell cotransfection of virion DNA from SV40/SEAP expressing Tip deletion virus along with linearized pSV40/GFP, replacement of the SEAP expression cassette with the GFP cassette via homologous recombination was performed.
Read the entire page →
From page 11391...
... The SV40/SEAP expression cassette was designed to contain restriction enzyme recognition sites not present in the HVS genome. AscI sites flanking the complete expression cassette and NotI sites flanking the SEAP gene were included to allow removal of either the entire expression cassette or the reporter gene only.
Read the entire page →
From page 11392...
... were SEAP-negative and GFPnegative, indicating that the virus in these wells was derived from self-ligation of the AscI-digested virus (Table 1~. The results clearly indicate that the SEAP reporter gene alone or the entire SV40/SEAP expression cassette can be removed directly from virion DNA and that it is possible to directly clone expression cassettes into the AscI sites of the recombinant virus.
Read the entire page →
From page 11393...
... Positive selection of SEAP or GFP aids in isolation of initial recombinant HVS and then provides in the purified recom binant the equally useful negative selection tool, loss of the reporter, for isolation of marker-rescued virus or other re combinants as desired. We are currently using these proce dures to isolate HVS strains with point mutations in STP and Tip and to isolate HVS recombinants capable of expressing antigens of other organisms for the purpose of vaccination.
Read the entire page →
From page 11394...
... 11394 Colloquium Paper: Duboise et al. Nicholas, J., Cameron, K
Read the entire page →

Key Terms

  • expression cassette
  • reporter gene
  • homologous recombination
  • herpesvirus saimiri
  • omk cells

  • recombinant virus
  • recombinant hvs
  • seap expression
  • limiting dilution
  • direct cloning

  • gene transfer
  • recombinant viruses
  • transfer vectors
  • deletion mutant
  • restriction enzyme

  • sites flanking
  • restriction sites
  • foreign gene
  • engineered alkaline
  • simian virus

  • cloning vector
  • green fluorescent
  • secreted engineered
  • fluorescent protein
  • cell culture

  • tyrosine kinase
  • alkaline phosphatase
  • bovine serum
  • human herpesvirus
  • expression cassettes


    • This material may be derived from roughly machine-read images, and so is provided only to facilitate research.
    More information on Chapter Skim is available.