Axonal degeneration of rat culured nerve introduced mutant aprata in cDNA

Koenig和我们鉴定了日本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用不能（AOA）的相关基因“aprataxin”。绝大多数EOCA-HA患者的aprataxin基因突变均为689插入T（689 ins T）。首先，我们通过PCR定点突变的方法获得了一个689 ins的T cDNA。其次，我们构建了一个同时含有野生型和689 ins T aprataxins的腺病毒载体。第三，以胎鼠背根为材料，建立了体外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抗体荧光染色和电镜观察感染腺病毒载体的神经的髓鞘形成和轴突出芽。构建的含野生型和689 ins T aprataxin基因的腺病毒载体感染培养神经后，神经髓鞘和轴突的变化无明显差异，应进一步获得含689 ins T的转基因小鼠。应分析这只小鼠的成熟神经。

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.