Promoter competition assay for analyzing gene regulation in joint tissue engineering.
We describe a new biochemical technique, "promoter competition assay," for examining the role of cis-acting DNA elements in tissue cultures. Recent advances in tissue engineering permit the culture of a variety of cells. Many tissues are engineered, however, without an appropriate understanding of molecular machinery that regulates gene expression and cellular growth. For elucidating the role of cis-acting regulatory elements in cellular differentiation and growth, we developed the promoter competition assay. This assay uses a transient transfer into cells of double-stranded DNA fragments consisting of cis-acting regulatory elements. The transferred DNA fragments act as a competitor and titrate the function of their genomic counterparts. Using synovial cells derived from a rheumatoid arthritis patient, we examined a role of NF-kappa B binding sites in the regulation of the expression of matrix metalloproteinase (MMP) genes. The results support a stimulatory role of NF-kappa B in transcriptional regulation of MMP-1 and MMP-13.
Biomedical Engineering Program, Department of Anatomy and Cell Biology, Indiana University, Indianapolis, Indiana 46202, USA.
Hui Bin
HB
Sun
George M
GM
Malacinski
Hiroki
H
Yokota
D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology; D001667:Binding, Competitive / Q000235:genetics; D002460:Cell Line; D002461:Cell Line, Transformed; D004247:DNA / Q000235:genetics / Q000502:physiology; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D007717:Knee / Q000473:pathology; D020782:Matrix Metalloproteinases / Q000096:biosynthesis; D016328:NF-kappa B / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D013314:Stress, Mechanical; D013583:Synovial Membrane / Q000378:metabolism*; D023822:Tissue Engineering / Q000379:methods*
D016328:NF-kappa B; D012333:RNA, Messenger; D004247:DNA; D020782:Matrix Metalloproteinases