| Tumor suppressor genes play an important role in the initiation and progression of human cancers. Positional cloning has been the classical approach in the isolation of tumor suppressor genes. A genomic interval of approximately one megabase at 8p22 has emerged as a possible site for a tumor suppressor gene, based on high rates of allelic loss in many different cancers of epithelial origin, and the presence of homozygous deletions found in two prostate cancers.; One objective of this study was to prepare a high-resolution physical map consisting of overlapping bacterial clones containing human genomic DNA across this region. A contig that completely spans the interval is presented here. The resulting physical map consists of 29 genomic clones and provides two-fold or greater coverage over much of the region. In addition, three new EST markers residing within the deletion interval, that are expressed in a human colorectal cancer cell line, were identified by searching the human gene map.; To utilize bacterial artificial chromosome (BAC) clones in biological studies, a novel method was devised of modifying these clones using cre recombinase to introduce a gene cassette into the loxP sequence that is present in the vector portion of the BAC clone. Cre-mediated integration is site-specific and thus maintains the integrity of the genomic insert. The success of this method depends on the use of a DNA construct, RETRObac, that contains the reporter marker GFP (green fluorescent protein) and the selectable marker neo (neomycin-phosphotransferase), but does not contain a bacterial origin of replication. Modified BAC clones show no signs of deletions or rearrangements and express genes that are contained in the genomic insert. The versatility associated with this retrofitting technique makes it a valuable resource in efforts to study biological effects of known genes, as well as in efforts to identify and analyze new genes and regulatory regions. |
PDF Full Text Request