Cftr

That oocytes and insects cells are incubated at lower temperatures, Denning et al. (6) examined the effect of incubating mammalian cells expressing AF508 CFTR at 26 C instead of 37 C. Now correct folding of the AF508 CFTR mutant was observed. Moreover, and like the situation with the insects cells and oocytes, cAMP-regulated chloride transport was observed in those cells expressing the mutant protein. Thus, we now know that the loss of a single phenylalanine residue at position 508 within the...

Generation of Mice of the Appropriate Genotype

The objective of the breeding program is to generate second generation pups that carry at least one copy of the SV40 transgene and are either non-CF (CFTR + +, or + -) or CF (CFTR - -). A male ImmortoMouse is placed with three female mice heterzygous for the S489X CFTR mutation in a microisolator cage. Multiple breeding cages are established to increase the number of first-generation offspring. When the female mice become pregnant, they are transferred to individual cages and are replaced with...

Cell Culture and Expansion

Ca2+- and Mg2+-free Hanks balanced salt solution that contains 0.05 trypsin and 0.53 mM EDTA (Gibco-BRL). 3. Collecting tubule culture media. 1 1 mix of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium (GIBCO-BRL) supplemented with 1.3 yg L sodium selenite, 1.3 yg L 3,3',5-triiodo-L-thyronine, 5 mg L insulin, 5 mg L transferrin, 25 yg L prostaglandin E1, 2.5 mM glutamine, 5 nM dexamethasone, 50000 U L nystatin, 50 mg L streptomycin, 30 mg L...

Mmzzm npe atp

Comparison of CFTR activation by rapid solution exchange and by caged ATP photolysis. (A) Trace shows activation of CFTR channels by rapid introduction of ATP, then by specific decaging of ATP-P3- 1-(2-nitrophenyl)ethyl ester (NPE-ATP). NPE-ATP does not induce activity until photolyzed by UV irradiation. Upward deflections indicate channel openings. Thin bars indicate rapid perfusion of ATP, filled bars perfusion of NPE-ATP, and stippled bars interruption of continuous perfusion....

Reconstitution of CFTR Protein into Liposomes from PFO

To study the structure and function of a purified membrane protein, it is essential that the detergent in which the protein is purified be exchanged for phospholipids. This exchange is a time- and concentration-dependent process. Typically, phospholipids are added to detergent-solubilized proteins and then the detergent is removed by dialysis or detergent adsorption resins 24,25 . Only when the detergent concentration decreases below a critical value critical micelle concentration or CMC can...