A study on an aberrant differentiation into chondrocytes in progressive ankylosis (ank/ank)

进行性强直(ANK/ANK)是一种常染色体隐性遗传性骨骼疾病。为了解ANK/ANK小鼠关节的精确病理变化，取ANK/ANK和ANK/ANK小鼠关节组织，石蜡包埋，HE染色切片。4周龄的ANK/ANK小鼠滑膜细胞向软骨细胞分化，滑膜增厚。在一只8周龄的小鼠中，增厚的滑膜部分钙化，关节软骨表面变得不规则。为了定位进行性强直的致病基因，我们利用F2纯合子小鼠与另一品系小鼠失血，分析了15号染色体遗传标记上的重组事件。在400个减数分裂中，我们在D15Mit130处发现了6个重组，重组最少。我们将Ank基因座定位在D15Mit130和D15Mit6之间1.25 cM的区域。为了缩小区域范围，我们计划利用BAC克隆和寡聚GT引物寻找多态标记，因为该区域没有更多的信息标记。我们利用抑制消减杂交技术(SSH)进行克隆。因此，我们目前获得了两个阳性克隆，它们在ANK/ANK小鼠的组织中表达低于未受影响的小鼠。为了进一步的研究，我们还将对cDNA进行全长延伸，并定位基因座。

Progressive ankylosis (ank/ank) is an autosomal recessive skeletal disorder in mice. To investigate the precise pathological changes in the joints of ank/ank, the joint tissues were removed from ank/+ and ank/ank mice, paraffin-embedded, and examined with H.E. stained sections. In a 4 week-old ank/ank mouse, the differentiation of synovial cells into chondrocytes was seen and the synovial membrane was thickened by the proliferation of the chondrocytes. In an 8 week-old mouse, the thickened synovial membranes were partially calcified and the surface of the articular cartilage became irregular.To map the causative gene of progressive ankylosis, we analyzed the recombination events at genetic markers in chromosome 15 using F2 homozygous mice bled with the other strain mice. Of 400 meioses, we found 6 recombinations at D15Mit130 for the minimum of recombination. We assigned the Ank locus to the 1.25 cM region between D15Mit130 and D15Mit6. To narrow down the region, we plan to find polymorphic markers with BAC clones and oligo GT primers because there is no more informative marker in the region.In parallel with the positional cloning, the subtraction cloning was carried out. We took advantage of suppression subtractive hybridization (SSH) for the cloning. As a result, we obtained, at the present time, two positive clones that are expressed less in the tissues of ank/ank mice than those of unaffected mice. For the further study we sill extend the cDNAs to the full length and map the gene loci.