Open AccessCFTR expression from a BAC carrying the complete human gene and associated regulatory elements
Author(s) -
Kotzamanis George,
Abdulrazzak Hassan,
GiffordGarner Jennifer,
Haussecker Pei Ling,
Cheung Wing,
GrillotCourvalin Catherine,
Harris Ann,
Kittas Christos,
Kotsinas Athanasios,
Gorgoulis Vassilis G.,
Huxley Clare
Publication year - 2009
Publication title -
journal of cellular and molecular medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.44
H-Index - 130
eISSN - 1582-4934
pISSN - 1582-1838
DOI - 10.1111/j.1582-4934.2008.00433.x
Subject(s) - biology , gene , bacterial artificial chromosome , gene expression , genetics , genomic dna , coding region , regulatory sequence , homologous recombination , microbiology and biotechnology , regulation of gene expression , genome
The use of genomic DNA rather than cDNA or mini‐gene constructs in gene therapy might be advantageous as these contain intronic and long‐range control elements vital for accurate expression. For gene therapy of cystic fibrosis though, no bacterial artificial chromosome (BAC), containing the whole CFTR gene is available. We have used Red homologous recombination to add a to a previously described vector to construct a new BAC vector with a 250.3‐kb insert containing the whole coding region of the CFTR gene along with 40.1 kb of DNA 5′ to the gene and 25 kb 3′ to the gene. This includes all the known control elements of the gene. We evaluated expression by RT‐PCR in CMT‐93 cells and showed that the gene is expressed both from integrated copies of the BAC and also from episomes carrying the oriP/EBNA‐1 element. Sequencing of the human CFTR mRNA from one clone showed that the BAC is functional and can generate correctly spliced mRNA in the mouse background. The BAC described here is the only CFTR genomic construct available on a convenient vector that can be readily used for gene expression studies or in vivo studies to test its potential application in gene therapy for cystic fibrosis.