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. W e have used this vector to deliver the cDNA for the cystic fibrosis tr ansmembrane conductance regulator (CFTR) to cells, both in vitro and i n vivo, complexed with cationic liposomes. 2. Human 293 kidney epithel ial 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 tr ansfected with pTrial10-CFTR2, but not pTrial10, demonstrated a cAMP-d ependent anion conductance, measured by fluorescence microscopy using a halide-sensitive probe, SPQ. 4. The CFTR-dependent, cAMP-sensitive c hloride secretory response in murine tracheal epithelium could be meas ured if the calcium-dependent chloride secretory process was first max imally stimulated with a mixture of the Ca2+-ATPase inhibitor, TBHQ, a nd 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 forskoli n in wild-type and CF tracheas was highly significantly different (P < 0.001), giving a CFTR-dependent current of 11.2 mu A cm(-2). 5. Trans fection of the airways with pTrial10-CFTR2, but not pTrial10, signific antly (P < 0.01) increased the CFTR-dependent chloride secretory curre nt in CF tracheas. The degree of correction was greater when intra-tra cheal installation rather than nasal insufflation was used to deliver the plasmids.