Develpment of a new method to revent amyloid formation in genetically engineered mice.

家族性淀粉样多发性神经病是一种常染色体显性遗传病，以周围神经和自主神经多发性神经病为特征。这种疾病是由甲状腺素运载蛋白（ttr）基因突变引起的，导致系统性淀粉样变性淀粉样蛋白形成过程包括ttr四聚体解离成单体、单体修饰、单体聚集、淀粉样蛋白原纤维形成以及血清淀粉样蛋白P组分（SAP）附着于淀粉样蛋白原纤维。为了阐明这些步骤中的相关因素并设计新的治疗方法，我们尝试用我们自己开发的Cre-突变型lox系统来培育一种可更换盒子的小鼠。我们构建了一个用于ES细胞同源重组的载体，它由DT-A、2.6kb的ttr同源区、lox71、PGK-neo、loxP、polyA、lox2272和7.9kb的ttr同源区组成。我们生产的嵌合体小鼠现在正在与雌性小鼠交配。为了分析肠道植物群的作用，我们从SPF小鼠转移肠道植物群 关于我们 e以及饲养在两个不同设施1和2中的常规小鼠与转基因小鼠。我们发现，淀粉样蛋白沉积在转基因小鼠的消化道从常规2，而不是从SPF和常规1，并在常规2条件下的共生体减少，伴随着增加的弱病原体，而保持。提示肠道植物群可影响淀粉样蛋白在消化道的沉积。另一方面，我们提出ttr分子中10位半胱氨酸残基之间的二硫键是单体聚集的先决条件。为了验证这种可能性，我们通过引入分别在第10位和第30位含有丝氨酸和甲硫氨酸残基的突变ttr基因来产生转基因小鼠。令人惊讶的是，我们在这些转基因小鼠中没有观察到淀粉样蛋白沉积，尽管在携带突变ttr基因的转基因小鼠中观察到淀粉样蛋白沉积，该突变ttr基因在30位携带甲硫氨酸。这些结果表明，位置10处的半胱氨酸残基对于单体彼此聚集将是重要的。我们还测试了Pepys等人开发的CPHPC是否可以从血清和淀粉样纤维中去除SAP。我们发现，使用携带人SAP基因Less的转基因小鼠，

Familial amyloidotic polyneuropathy is an autosomal dominant disorder characterized by peripheral and sutonomic polyneuropathy. This disease was caused by the mutation in the transthyretin (ttr) gene, leading to systemic amyloidosis Amyloidogenic processes include dissociation of ttr tetramers into monomers, modification of monomers, aggregation of monomers, formation of amyloid fibrils, and attachment of serum amyloid P component (SAP) to amyloid fibrils. To elucidate the factors involved in these steps and to devise a new way of treatment, we tried to develop a casette exchnageable mouse using Cre-mutant lox system which was developed by ourselves. We made a vector for homologous recombination in ES cells, consisting of DT-A, 2.6 kb of homologous region of ttr, lox71, PGK-neo, loxP, poly A, lox2272, and 7.9kb homologous region of ttr. We produce chimeric mice that are now mating with female mice. To analyze the effects of intestinal flora, we transferred intestinal flora from SPF mic … More e as well as conventional mice housed in two different facilities 1 and 2 to transgenic mice. We found that amyloid was deposited in alimentary tract of transgenic mice transferred from conventional 2, but not from SPF and conventional 1, and that in conventional 2 condition symbiotes were decreased accompanied by an increase of weak pathogens while keeping. These suggest that intestinal flora can affect the amyloid deposition in alimentary tract. On the other hand, it is proposed that disulfide bond between cystein residues at position 10 in ttr molecule is prerequisite for aggregation of monomer. To test this possibility, we produce transgenic mice by introducing mutant ttr gene containing serine and methionine residues at position 10 and 30, respectively. Surprisingly, we could not observe amyloid deposition in these transgenic mice, although amyloid deposition was observed in transgenic mice carrying a mutant ttr gene carrying methionine at position 30. These results suggest that cystein residue at position 10 will be important for monomers to aggregate each other. We also tested whether CPHPC developed by Pepys et al. can remove SAP from serum and amyloid fibrils. We found that SAP was effectively removed from serum and amyloid deposits using transgenic mice carrying human SAP gene Less