High-efficiency transfer of cystic fibrosis transmembrane conductance regulator cDNA into cystic fibrosis airway cells in culture using lactosylated polylysine as a vector.
To find more efficient vectors for the transfer of CFTR cDNA, lactosylated polylysine was explored for transfer into airway epithelial cells in primary culture. The efficacy and high efficiency of transfection were shown by several criteria: expression of both mRNA and protein for CFTR and the functional correction of the Cl- channel activity. Using specific combinations of agents to enhance the transfection, an efficiency of 90% was obtained as detected by in situ hybridization with digoxigenin-labeled probes generated against exon 14 of CFTR. The highest efficiency was observed by adding E5CA peptide (10 microg) and 5% glycerol to the transfection mixture. The degree of transfection could be controlled by the enhancing agents, thus modulating the efficiency of transfection. The highest level of transfection efficiency is equivalent to that reported for viral vectors. None of the agents or their combinations in the concentrations used were cytotoxic to the primary cells. Antibody pAb3145 was used to detect the expression of the CFTR protein in the cells. When an N-terminal GFP-CFTR fusion gene was used to transfect the CF cells a functional correction of the CFTR Cl- channel was detected by patch-clamp electrophysiology. The high efficiency of CFTR gene transfer with lactosylated polylysine leads to the conclusion that lactosylated polylysine is a promising vector to transfer the CFTR gene into human airway cells in culture.
Kollen
W J
WJ
Department of Pediatrics, University of Pennsylvania School of Medicine, The Children's Hospital of Philadelphia, 19104-4318, USA.
Mulberg
A E
AE
Wei
X
X
Sugita
M
M
Raghuram
V
V
Wang
J
J
Foskett
J K
JK
Glick
M C
MC
Scanlin
T F
TF
D001483:Base Sequence; D002461:Cell Line, Transformed; D003550:Cystic Fibrosis / Q000473:pathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary; D004337:Drug Carriers; D018014:Gene Transfer Techniques; D011107:Polylysine / Q000008:administration & dosage*; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D004337:Drug Carriers; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011107:Polylysine