Genomic studies of gene expression: regulation of the Wilson disease gene.
Bacterial artificial chromosomes (BACs) have many advantages over other large-insert cloning vectors and have been used for a variety of genetic applications, including the final contigs of the human genome. We describe the utilization of a BAC construct to study gene regulation in a tissue culture-based system, using a 170-kb clone containing the entire Wilson disease (WND) locus as a model. A second BAC construct that lacked a putative negatively regulating promoter sequence was created. A nonviral method of gene delivery was applied to transfect three human cell lines stably with each construct. Our results show correct WND gene expression from the recombinant locus and quantification revealed significantly increased expression from the clone lacking the negative regulator. Comparison with conventional methods confirms the reliability of the genomic approach for thorough examination of gene expression. This experimental system illustrates the potential of BAC clones in genomic gene expression studies, new gene therapy strategies, and validation of potential molecular targets for drug discovery.
Bochukova
Elena G
EG
Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
Jefferson
Andrew
A
Francis
Michael J
MJ
Monaco
Anthony P
AP
D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000235:genetics*; D027682:Cation Transport Proteins / Q000096:biosynthesis / Q000235:genetics*; D045744:Cell Line, Tumor; D022202:Chromosomes, Artificial, Bacterial; D000073840:Copper-Transporting ATPases; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D005809:Genes, Regulator / Q000235:genetics; D023281:Genomics / Q000379:methods*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017384:Sequence Deletion; D014162:Transfection
D027682:Cation Transport Proteins; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases