Previous Article | Next Article ![]()
Journal of Virology, November 1998, p. 8861-8872, Vol. 72, No. 11
Cystic Fibrosis/Pulmonary Research and
Treatment Center and Department of Medicine, University of North
Carolina at Chapel Hill, Chapel Hill, North
Carolina,1 and
Center for Molecular
Genetics and Department of Pediatrics, University of California
Received 24 November 1997/Accepted 14 July 1998
To study retroviral gene transfer to airway epithelia, we used a
transient transfection technique to generate high titers (~109 infectious units/ml after concentration) of murine
leukemia virus (MuLV)-derived vectors pseudotyped with the vesicular
stomatitis virus envelope glycoprotein (VSV-G). Transformed (CFT1) and
primary airway epithelial cells were efficiently transduced by a
VSV-G-pseudotyped lacZ vector (HIT-LZ) in vitro. CFT1 cells
and primary cystic fibrosis (CF) airway cell monolayers infected with a
vector (HIT-LCFSN) containing human CF transmembrane conductance
regulator (CFTR) in the absence of selection expressed CFTR, as
assessed by Western blot analysis, and exhibited functional correction
of CFTR-mediated Cl
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effect of Host Modification and Age on Airway
Epithelial Gene Transfer Mediated by a Murine Leukemia
Virus-Derived Vector
San
Diego, San Diego, California2
secretion. In vitro studies of
persistence suggested that pseudotransduction was not a significant
problem with our vector preparations. In a sulfur dioxide
(SO2) inhalational injury model, bromodeoxyuridine (BrdU)
incorporation rates were measured and found to exceed 50% in
SO2-injured murine tracheal epithelium. HIT-LZ vector
(multiplicity of infection of ~10) instilled into the
SO2-injured tracheas of anesthetized mice transduced 6.1% ± 1.3% of superficial airway cells in tracheas of weanling mice (3 to
4 weeks old; n = 10), compared to 1.4 ± 0.9% in
mice 5 weeks of age (n = 4) and 0.2% in mice older
than 6 weeks (n = 15). No evidence for gene transfer following delivery of HIT-LZ to tracheas of either weanling or older
mice not injured with SO2 was detected. Because only a
small fraction of BrdU-labeled airway cells were transduced, we
examined the stability of the vector. No significant loss of vector
infectivity over intervals (2 h) paralleling those of in vivo protocols
was detected in in vitro assays using CFT1 cells. In summary,
high-titer vectors permitted complementation of defective CFTR-mediated
Cl
transport in CF airway cells in vitro without
selection and demonstrated that the age of the animal appeared to be a
major factor affecting in vivo retroviral transduction efficiency.
*
Corresponding author. Mailing address: Cystic
Fibrosis/Pulmonary Research and Treatment Center, CB 7248, 7123A
Thurston Bowles Bldg., University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599-7248. Phone: (919) 966-7052. Fax: (919) 966-7524. E-mail: gdoc{at}med.unc.edu.
This article has been cited by other articles:
| J. Bacteriol. | Mol. Cell. Biol. | Microbiol. Mol. Biol. Rev. |
|---|
| Clin. Vaccine Immunol. | ALL ASM JOURNALS |
|---|