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白蛋白血症（EOCA-HA）和葡萄牙小脑性共济失调伴眼失用（AOA）的相关基因“aprataxin”。大多数EOCA-HA患者的aprataxin基因突变为689插入T（689 in T）纯合。首先，利用PCR技术进行定点诱变，获得689个片段的T cDNA。其次，构建了含有野生型和689型T - aprataxins的腺病毒载体。第三，用胎鼠背根培养大鼠周围神经。用构建的腺病毒载体感染培养的神经，用抗po抗体和电镜观察感染构建的腺病毒载体培养的神经的髓鞘形成和轴突发芽情况。在培养的神经感染含有野生和689基因的腺病毒载体后，观察到髓鞘和轴突的变化没有差异。进一步，应获得含有689 in T的转基因小鼠。该小鼠的成熟神经应进行分析。

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.