Chloride secretion in the trachea of null cystic fibrosis mice: the effects of transfection with pTrial10-CFTR2.
1. An improved novel plasmid backbone, pTrial10, has been developed. We have used this vector to deliver the cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR) to cells, both in vitro and in vivo, complexed with cationic liposomes. 2. Human 293 kidney epithelial cells (HEK 293) showed expression of an immunoprecipitable 165 kDa protein corresponding to CFTR when transfected in vitro with pTrial10-CFTR2, but not when the vector pTrial10 was used. 3. HEK 293 cells transfected with pTrial10-CFTR2, but not pTrial10, demonstrated a cAMP-dependent anion conductance, measured by fluorescence microscopy using a halide-sensitive probe, SPQ. 4. The CFTR-dependent, cAMP-sensitive chloride secretory response in murine tracheal epithelium could be measured if the calcium-dependent chloride secretory process was first maximally stimulated with a mixture of the Ca(2+)-ATPase inhibitor, TBHQ, and the calcium ionophore, A23187. With these conditions wild-type and CF-null (transgenic animals in which the cystic fibrosis (CF) gene has been disrupted so that no CFTR is produced) murine tracheas could be distinguished. The difference between the current elicited by forskolin in wild-type and CF tracheas was highly significantly different (P < 0.001), giving a CFTR-dependent current of 11.2 microA cm-2. 5. Transfection of the airways with pTrial10-CFTR2, but not pTrial10, significantly (P < 0.01) increased the CFTR-dependent chloride secretory current in CF tracheas. The degree of correction was greater when intra-tracheal installation rather than nasal insufflation was used to deliver the plasmids.
MacVinish
L J
LJ
Department of Pharmacology, University of Cambridge, UK.
Gill
D R
DR
Hyde
S C
SC
Mofford
K A
KA
Evans
M J
MJ
Higgins
C F
CF
Colledge
W H
WH
Huang
L
L
Sorgi
F
F
Ratcliff
R
R
Cuthbert
A W
AW
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001358:Avian Sarcoma Viruses / Q000235:genetics; D000001:Calcimycin / Q000494:pharmacology; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000628:therapy; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004232:Diuretics / Q000494:pharmacology; D015316:Genetic Therapy; D006367:HeLa Cells; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D018181:Mice, Inbred CFTR; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014132:Trachea / Q000378:metabolism*; D014162:Transfection
D000975:Antioxidants; C505032:CFTR protein, human; D018118:Chloride Channels; D002712:Chlorides; D004232:Diuretics; D006873:Hydroquinones; D007476:Ionophores; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000001:Calcimycin; D000584:Amiloride; C018855:2-tert-butylhydroquinone; D000242:Cyclic AMP; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid