title: Concordant activity of transgene expression cassettes inserted into E1, E3 and E4 cloning sites in the adenovirus genome.
abstract: BACKGROUND
  Expression cassettes can be inserted at several positions into recombinant adenoviral genomes but the implications of this choice for transgene expression level have not been determined. Knowledge of the relative expression levels of transgenes inserted at different sites in the adenoviral genome is of particular significance for transgene expression monitoring approaches that rely on the concordant expression of a marker transgene inserted elsewhere in the viral genome.
  
  
  METHODS
  Three expression cassettes, each comprising a cytomegalovirus promoter driving one of three marker peptides [serum carcinoembryonic antigen (sCEA), beta subunit of human chorionic gonadotropin (betahCG) or human sodium iodide symporter (hNIS)], were inserted into E1, E3 or E4 cloning sites in a recombinant adenoviral vector backbone. High titer stocks of bicistronic adenoviral vectors coding for combinations of marker peptides were prepared. A panel of human cells of various lineages was infected with the vectors and expression ratios of the transgene-encoded proteins were analysed. Serum levels of the soluble proteins and hepatic uptake of radioactive iodine were also compared in vivo in nude rats after intravenous vector infusion.
  
  
  RESULTS
  High concordance of expression between the inserted transgenes was observed in all of the bicistronic vectors irrespective of whether the expression cassettes were placed in the E1, E3 or E4 regions. Concordance was maintained across multiple cell lineages. In vivo, in athymic rats, blood and urine levels of betahCG were highly concordant with serum levels of sCEA at all timepoints after intravenous infusion of the bicistronic vectors encoding both of these soluble markers. Hepatic radioiodine uptake was concordant with serum CEA concentration in mice infused with a bicistronic vector expressing CEA and NIS.
  
  
  CONCLUSIONS
  The expression level of a given transgene in an adenoviral vector genome can be accurately and quantitatively inferred from the expression of a marker protein encoded by a second transgene inserted elsewhere in the vector genome.
authors.affiliation: Department of Molecular Medicine, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
authors.forename: Linh
authors.initials: L
authors.lastname: Pham
authors.forename: Takafumi
authors.initials: T
authors.lastname: Nakamura
authors.forename: A
authors.initials: A
authors.lastname: Gabriela Rosales
authors.forename: Stephanie K
authors.initials: SK
authors.lastname: Carlson
authors.forename: Kent R
authors.initials: KR
authors.lastname: Bailey
authors.forename: Kah-Whye
authors.initials: KW
authors.lastname: Peng
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Russell
mesh_terms: D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002272:Carcinoembryonic Antigen / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics / Q000378:metabolism; D005260:Female; D015967:Gene Expression Regulation, Viral; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D016679:Genome, Viral; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D051381:Rats; D011923:Rats, Nude; D019076:Transgenes
chemical_list: D002272:Carcinoembryonic Antigen; D018997:Chorionic Gonadotropin, beta Subunit, Human