Gene Candidates for Embryonic Lethals in the The Mouse T-complex

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From a sequence tagged site (STS)/Bacterial Artificial Chromosome based physical map in mouse t-complex region 43 BACs were representing a minimal tiling path were selected, sequenced, annotated and deposited into the public databases. Based on the physical map the tw5 embryonic lethal locus mutation was refined to a single, 175 Kb BAC. Histological studies have demonstrated that mice homozygous for tw5 die at the gastrulation stage. The candidate BAC rescues the tw5 lethality, restoring viability. Our collaborator Dr. K. Abe is currently focusing on the search for the gene mutated in the tw5 embryonic lethality. Currently, we have focused our attention on the mouse homolog of human ADAMTS10 gene, which maps to the interval. Several metalloproteases play critical roles in cell-cell signal transduction. ADAMTS-10 gene, a zinc metalloendopeptidase with thrombospondin domains, is a potential candidate for important functions during development. Recently ADAMTS10 was found to be a candidate gene for Weill-Marchesani syndrome (MIM#277600). Previously we found that mouse ADAMTS-10 is alternatively spliced in a tissue-specific manner and encodes protein isoforms that either include or exclude the thrombospondin domains by truncation of the open reading frame. We have cloned and characterized 3 major isoforms from embryo, kidney and lung, and find that the embryo, for example, expresses an isoform that has a truncated translation product. The truncated embryonic and full-length kidney isoforms were transfected into 293 cells. We find that both isoforms are stably expressed and localize to the cytoplasm, as determined by immunocytochemistry. Rabbit polyclonal antibodies were generated targeted to epitopes that could distinguish between different isoforms and are being purified and characterized for further use to define the protein function.