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Adenovirus-mediated overexpression of murine cyclic nucleotide phosphodiesterase 3B

Ahmad, Faiyaz; Härndahl, Linda LU ; Tang, Yan; Stenson, Lena LU and Manganiello, Vincent C (2005) In Methods in molecular biology (Clifton, N.J.) 307. p.93-107
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Chapter in Book/Report/Conference proceeding
publication status
published
subject
keywords
PDE3B, phosphorylation, adenovirus purification, real-time polymerase chain reaction, HEK 293 cells, insulin release
in
Methods in molecular biology (Clifton, N.J.)
volume
307
pages
93 - 107
publisher
Humana Press
external identifiers
  • pmid:15988058
  • scopus:24644477766
ISSN
1064-3745
1940-6029
DOI
10.1385/1-59259-839-0:093
language
English
LU publication?
yes
id
4312a7b6-0dc4-4d74-972d-30de24666161 (old id 1132243)
date added to LUP
2008-06-30 11:34:37
date last changed
2017-01-01 05:00:32
@inbook{4312a7b6-0dc4-4d74-972d-30de24666161,
  abstract     = {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.},
  author       = {Ahmad, Faiyaz and Härndahl, Linda and Tang, Yan and Stenson, Lena and Manganiello, Vincent C},
  issn         = {1064-3745},
  keyword      = {PDE3B,phosphorylation,adenovirus purification,real-time polymerase chain reaction,HEK 293 cells,insulin release},
  language     = {eng},
  pages        = {93--107},
  publisher    = {Humana Press},
  series       = {Methods in molecular biology (Clifton, N.J.)},
  title        = {Adenovirus-mediated overexpression of murine cyclic nucleotide phosphodiesterase 3B},
  url          = {http://dx.doi.org/10.1385/1-59259-839-0:093},
  volume       = {307},
  year         = {2005},
}