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

从小鼠t-复合体区域中基于序列标记位点（STS）/细菌人工染色体的物理图谱中，选择43个BAC代表最小平铺路径，测序，注释并存入公共数据库。基于物理图谱，将tw 5胚胎致死基因座突变细化为单个175 Kb BAC。组织学研究表明，tw 5纯合子小鼠在原肠胚形成阶段死亡。候选BAC挽救了tw 5的致死率，恢复了活力。我们的合作者K博士安倍目前正集中精力寻找tw 5胚胎致死突变基因。 目前，我们的注意力集中在人类ADAMTS 10基因的小鼠同源物上，该同源物定位于间隔。几种金属蛋白酶在细胞-细胞信号转导中起关键作用。ADAMTS-10基因是一种具有血小板反应蛋白结构域的锌金属内肽酶，是发育过程中重要功能的潜在候选基因。最近发现ADAMTS 10是Weill-Marchesani综合征的候选基因（MIM#277600）。以前，我们发现，小鼠ADAMTS-10是选择性剪接的组织特异性的方式和编码的蛋白质亚型，包括或排除血小板反应蛋白结构域的开放阅读框的截断。我们已经克隆并表征了来自胚胎、肾和肺的3种主要同种型，并且发现例如胚胎表达具有截短的翻译产物的同种型。将截短的胚胎和全长肾同种型转染到293细胞中。我们发现，这两种亚型的稳定表达和本地化的细胞质，免疫细胞化学测定。产生了靶向表位的兔多克隆抗体，这些表位可以区分不同的同种型，并且正在进行纯化和表征以进一步用于定义蛋白质功能。

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.