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

从小鼠t-复合体区域基于序列标记位点(STS)/细菌人工染色体的物理图谱中，选择43个代表最小平铺路径的BAC，对其进行测序、注释并存储到公共数据库中。根据物理图谱，TW5胚胎致死位点突变被提炼成一个175kb的BAC。组织学研究表明，TW5纯合子小鼠在原肠发育阶段死亡。候选人BAC拯救了TW5的致命性，恢复了生存能力。我们的合作者K.Abe博士目前正专注于寻找在TW5胚胎致死中突变的基因。目前，我们的注意力集中在人ADAMTS10基因的小鼠同源基因上，该基因映射到区间。几种金属蛋白酶在细胞间的信号转导中起着关键作用。ADAMTS-10基因是一种含有凝血酶反应蛋白结构域的锌金属内肽酶，在发育过程中可能具有重要的功能。最近发现ADAMTS10是Weill-Marchesani综合征(MIM#277600)的候选基因。以前，我们发现小鼠ADAMTS-10以组织特异性的方式选择性剪接，并通过截断开放阅读框来编码包括或排除凝血反应蛋白结构域的蛋白质异构体。我们已经克隆并鉴定了胚胎、肾脏和肺的三种主要亚型，并发现例如胚胎表达的一种亚型具有截短的翻译产物。将截短的胚胎和全长肾脏亚型分别导入293细胞。我们发现这两种异构体都稳定表达，并定位于细胞质，通过免疫细胞化学确定。针对能够区分不同异构体的表位产生了兔多克隆抗体，并正在进行纯化和鉴定，以便进一步用于确定蛋白质的功能。

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.