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性疾病，以外周和眼上型多发性神经病为特征。全身性淀粉样变性的发病过程包括TTR四聚体的解离、单体的修饰、单体的聚集、淀粉样纤维的形成以及血清淀粉样蛋白P组分(SAP)与淀粉样纤维的附着。为了阐明这些步骤中涉及的因素，并设计出一种新的治疗方法，我们试图利用我们自己开发的Cre突变的lox系统来培育一只干酪可交换的小鼠。我们构建了一个ES细胞同源重组载体，该载体由DT-A、2.6kb的TTR同源区、lox71、PGK-neo、loxP、polA、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。为了分析肠道菌群的影响，我们从SPF小鼠…中转移了肠道菌群。与传统小鼠一样，更多的e小鼠被安置在两个不同的设施1和2中，以获得转基因小鼠。我们发现常规2转基因小鼠的消化道中有淀粉样蛋白沉积，而SPF和常规1转基因小鼠则没有，并且在常规2条件下，共生体减少，同时伴随着弱病原菌的增加。提示肠道菌群可影响消化道淀粉样蛋白的沉积。另一方面，认为TTR10位半胱氨酸残基之间的二硫键是单体聚集的必要条件。为了测试这种可能性，我们通过在第10位和第30位分别引入含有丝氨酸和蛋氨酸残基的突变TTR基因来产生转基因小鼠。令人惊讶的是，我们没有在这些转基因小鼠中观察到淀粉样蛋白的沉积，尽管在第30位携带蛋氨酸的突变TTR基因的转基因小鼠中观察到了淀粉样蛋白的沉积。这些结果表明，第10位的半胱氨酸残基对单体相互聚集很重要。我们还测试了CPHPC是否由Pepys等人开发。能去除血清和淀粉样蛋白纤维中的SAP。我们发现，使用携带较少人SAP基因的转基因小鼠，SAP可以有效地从血清和淀粉样沉淀物中去除

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