Axonal degeneration of rat culured nerve introduced mutant aprata in cDNA

Koenig和我们在日本发现了早发性小脑性共济失调伴低蛋白血症(EOCA-HA)和葡萄牙的小脑性共济失调伴眼球失用(AOA)的相关基因“aprataxin”。大多数EOCA-HA患者aprataxin基因突变表现为689个插入T(689 Ins T)纯合。首先，我们在聚合酶链式反应的基础上，通过定点突变获得了689个INS-T基因。其次，我们构建了含有野生型和689INS Taprataxins的重组腺病毒载体。第三，我们用胎鼠背根制作了培养的大鼠周围神经。用构建的腺病毒载体感染培养的神经，用抗Po抗体和电子显微镜进行免疫组织化学染色，观察神经的髓鞘形成和轴突萌发。含有野生型和689INS Taprataxin基因的重组腺病毒载体感染培养的神经后，髓鞘形成和轴突变化无明显差异，进一步获得含有689INS T的转基因小鼠。应该对这只小鼠成熟的神经进行分析。

Koenig and we identified a responsible gene "aprataxin" for early-onset cerebellar ataxia with hypoalbuminemia(EOCA-HA) in Japan and cerebellar ataxia with ocular apraxia(AOA) in Portugal. The mutation of aprataxin gene in most EOCA-HA patients showed 689 insertion T(689 ins T) homozygously. First, we made a 689 ins T cDNA by site-directed mutagenesis bases on PCR. Second, we made a constructed adeno virus vector contained both wild and 689 ins T aprataxins. Third, we made a cultured rat peripheral nerve using dorsal root of fetal rat. Those cultured nerves were infected by constructed adeno virus vectors, Myelination and axonal sprouting of cultured nerves infected constructed adeno virus vector were observed by immunohistochenistory using anti-Po antibody and electron microscopy. No differences of myelination and axonal changes were observed in cultured nerves infected constructed adeno virus vector containing both a wild and 689 ins T aprataxin gene.Further more, transgenic mouse containing 689 ins T should be obtained. Matured nerve of this mouse should be analyzed.