Production of a transgenic mouse model of familial amyloidotic polyneuropathy.

家族性淀粉样多发性神经病是一种常染色体显性遗传疾病，其特征是淀粉样纤维的细胞外沉积和显著的外周神经和自主神经受累。这种淀粉样蛋白原蛋白主要由甲状腺素运载蛋白（TTR）组成，在FAP型中，第30位的缬氨酸被甲硫氨酸取代。I.为了分析淀粉样蛋白沉积的病理过程和突变TTR基因以外的因素，我们将克隆的人突变TTR基因显微注射到C57 BL/6小鼠的受精卵中，制备了转基因小鼠。为了在转基因小鼠中产生大量的变体TTR，我们制备了两种构建体。一个是含有约600个碱基对（bp）的上游区和完整的人突变TTR基因的7.6-hTTR（kb）片段（0.6-hTTR 30）。另一个是7.8kb片段，其中启动子区被小鼠金属硫蛋白-I基因（MT-hTTR 30）的启动子区所取代。我们分别生产了9只和5只转基因小鼠。两个品系之间的人突变TTR血清浓度没有显著差异，其范围为0.2至5.0/mg/dl。然而，从每个基因的断裂特异性f判断，MT-hTTR 30中由人TTR（hTTR 4）组成的同源四聚体的实际量比0.6-hTTR 30中高300倍。MT-hTTR 30细胞系消化道粘膜下层和肾小球内有淀粉样物质沉积，而0.6-hTTR 30细胞系无淀粉样物质沉积。这些结果表明hTTR 4的存在对于淀粉样蛋白沉积是重要的。但这些结果不能排除脉络丛中hTTR基因的表达对淀粉样蛋白沉积的重要性。我们正在尝试通过显微注射含有约6.0kb上游区的人突变TTR基因来制备转基因小鼠。

Familial amyloidotic polyneuropathy is an autosomal dominant disorder which is characterized by extracellular deposition of amyloid fibrils and by prominent peripheral and autonomic nerve involvement. This amyloid prothein is mainyl composed of transthyretin (TTR) with a substitution of methionine for valine at position 30 in the FAP type. I. This amino acid substitution is thought to lead to amyloid deposition.In order ot analyze the pathological process of amyloid deposition and the factor(s) other than mutant TTR gene, we have produced transgenic mice by microinjecting the cloned human mutant TTR gene into fertilized eggs of C57BL/6 mice. In order to produce large quantities of variant TTR in transgenic mice, we prepared two constructs. One is the 7.6-kilobase (kb) fragment (0.6-hTTR30) containing about 600-base pair (bp) upstream region and entire human mutant TTR gene. The other is the 7.8 kb fragment in which the promotor region was replaced with that of the mouse metallotheionein-I gene (MT-hTTR30). We produced 9 and 5 transgenic mice, respectively. There was no significant difference in serum concentrations of human mutant TTR between two lines and they ranged from 0.2 tof 5.0/mg/dl. However, the actual amount of homo-tetramers composed of human TTR (hTTR4) was 300 times higher in MT-hTTR30 than in 0.6-hTTR30 as judged from the fissue specificityf of each gene. Amyloid deposition was observed in the submucosa of alimentary tract and renal glomeruli of MT-hTTR30 line but not of 0.6-hTTR30 line. These results suggest that the presence of hTTR4 is important for amyloid deposition. But the possibility that the Xpression of hTTR gene in the choroid plexus is important for amyloid deposition cannot be ruled out from these results. We are now atempting ot produce transgenic mice by microinjecting the human mutant TTR gene containg about 6.0 kb upstream region.