Functional human CFTR produced by a stable minichromosome.
Artificial chromosomes have been claimed to be the ideal vector for gene therapy, but their use has been hampered by an inability to produce stable and well designed molecules. We have used a structurally defined minichromosome to clone the human cystic fybrosis transmembrane conductance regulator (CFTR) locus. To guarantee the presence of the proper regulatory elements, we used the 320 kb yeast artificial chromosome (YAC) 37AB12 with the intact CFTR gene and upstream sequences. The resulting minichromosome was analyzed for the presence of the entire CFTR gene and for its functional activity by molecular and functional methods. We have identified clones showing the presence of both the transcript and the CFTR protein. Moreover, in the same clones, a chloride secretory response to cAMP was detected. Mitotic and molecular stability after prolonged growth without selection demonstrated that the constructs were stable. This is the first example of a structurally known minichromosome made to contain an active therapeutic gene.
Auriche
Cristina
C
Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartmento di Biologia Cellulare e dello Sviluppo, University of Rome La Sapienza, Italy.
Carpani
Daniela
D
Conese
Massimo
M
Caci
Emanuela
E
Zegarra-Moran
Olga
O
Donini
Pierluigi
P
Ascenzioni
Fiorentina
F
D000818:Animals; D016466:CHO Cells; D002713:Chlorine / Q000378:metabolism; D002875:Chromosomes / Q000378:metabolism*; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis* / Q000235:genetics*; D005821:Genetic Techniques; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D002713:Chlorine; D000242:Cyclic AMP; D001616:beta-Galactosidase