CFTR expression and activity from the human CFTR locus in BAC vectors, with regulatory regions, isolated by a single-step procedure.
We have assembled two BAC vectors containing a single fragment spanning the entire CFTR locus and including the upstream and downstream regions. The two vectors differ in size of the upstream region, and were recovered in Escherichia coli, with intact BAC DNAs prepared for structural and functional analyses. Sequence analysis allowed precise mapping of the inserts. We show that the CFTR gene was wild type and is categorized as the most frequent haplotype in Caucasian populations, identified by the following polymorphisms: (GATT)₇ in intron 6a; (TG)₁₁T₇ in intron 8; V470 at position 470. CFTR expression and activity were analyzed in model cells by RT-PCR, quantitative real-time PCR, western blotting, indirect immunofluorescence and electrophysiological methods, which show the presence of an active CFTR Cl ⁻ channel. Finally, and supporting the hypothesis that CFTR functions as a receptor for Pseudomonas aeruginosa, we show that CFTR-expressing cells internalized more bacteria than parental cells that do not express CFTR. Overall, these data demonstrate that the BAC vectors contain a functional CFTR fragment and have unique features, including derivation from a single fragment, availability of a detailed genomic map and the possibility to use standard extraction procedures for BAC DNA preparations.
Auriche
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Dipartimento di Biologia Cellulare e dello Sviluppo, Sapienza Università di Roma, Roma, Italy.
Di Domenico
E G
EG
Pierandrei
S
S
Lucarelli
M
M
Castellani
S
S
Conese
M
M
Melani
R
R
Zegarra-Moran
O
O
Ascenzioni
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F
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D006801:Humans; D007438:Introns / Q000235:genetics*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator