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Adenovirus-mediated overexpression of murine cyclic nucleotide phosphodiesterase 3B

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Author

  • Faiyaz Ahmad
  • Linda Härndahl
  • Yan Tang
  • Lena Stenson
  • Vincent C Manganiello

Summary, in English

To construct the recombinant adenovirus vector containing the cDNA for recombinant mouse cyclic nucleotide phosphodiesterase 3B (mPDE3B), the cDNA for mPDE3B was subcloned into pACCMV.pLpA. Subsequently, this recombinant plasmid, pACCMV.mPDE3B, was cotransfected with pJM17 plasmid containing the adenoviral genome into 293 human embryonic kidney cells, and the replication-deficient adenovirus AdCMV.mPDE3B was generated via homologous recombination. Large-scale preparation of adenovirus yielded 10(11)-10(13) viral particles/mL and could be quantitated by real-time polymerase chain reaction using iCycler (Bio-Rad). Efficiency of gene transfer was assessed by infecting FDCP2 or H4IIE cells with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 75% of cells were infected. Expression of mPDE3B in H4IIE hepatoma cells, FDCP2 hematopoietic cells, and beta-cells from isolated pancreatic islets was detected by Western blot analysis. In lysates from FDCP2 cells and H4IIE hepatoma cells infected with recombinant adenoviral mPDE3B constructs, mPDE3B activity was increased 10- to 30-fold compared with the activity in lysates from cells infected with beta-gal adenovirus. Stimulation of FDCP2 cells infected with mPDE3B adenovirus with insulin (100 nM, 10 min) resulted in an approx 1.7-fold increase in endogenous PDE3B and recombinant wild-type PDE3B activities. Infection of rat pancreatic islets resulted in a 5- to 10-fold increase in PDE3B expression and activity and subsequent blunting of insulin secretion. Thus, adenovirus-mediated gene transfer is effective for studying expression and regulation of recombinant PDE3 in insulin-responsive cells as well as insulin-secreting cells.

Publishing year

2005

Language

English

Pages

93-107

Publication/Series

Methods in molecular biology (Clifton, N.J.)

Volume

307

Document type

Book chapter

Publisher

Humana Press

Topic

  • Endocrinology and Diabetes

Keywords

  • PDE3B
  • phosphorylation
  • adenovirus purification
  • real-time polymerase chain reaction
  • HEK 293 cells
  • insulin release

Status

Published

Research group

  • Insulin Signal Transduction

ISBN/ISSN/Other

  • ISSN: 1064-3745
  • ISSN: 1940-6029