A study of the molecular mechanism and the gene therapy for congenital myotonic dystrophy.

1、先天性强直性肌营养不良症的产前诊断方法建立了非同位素PCR和Southern印迹法检测肌紧张素/蛋白激酶基因CTG重复序列异常的方法，用于诊断先天性强直性肌营养不良症。对该基因的CTG重复序列和侧翼标记进行了产前诊断分析。主要应用于脆性X综合征和亨廷顿病的基因异常分析，其基因异常机制相似。2、肌紧张素/蛋白激酶基因在亨廷顿病淋巴母细胞中的表达分析采用PCR技术分析了淋巴母细胞中肌紧张素/蛋白激酶mRNA的表达。建立了竞争PCR技术，比较正常和先天性强直性肌营养不良症样本之间的mRNA量。肌紧张素/蛋白激酶基因的表达水平在正常和疾病样本中没有差异。肌紧张素/蛋白激酶基因的mRNA量在淋巴母细胞中非常低。我们将尝试用肌肉进行这项研究。3、肌紧张素/蛋白激酶蛋白在大肠杆菌中的表达我们尝试用大肠杆菌表达系统表达肌紧张素/蛋白激酶蛋白。从人和小鼠中制备了全长的肌紧张素/蛋白激酶cDNA，并将其亚克隆到pRSET载体中，在大肠杆菌中表达。在该系统中没有明确检测到肌紧张素/蛋白激酶蛋白。我们将尝试使用该基因的部分片段或使用其他表达系统。

1, An method of the prenatal diagnosis for congenital myotonic dystrophy.We established the non-radioisotope PCR and Southern blot method to analyze CTG repeat size abnormality of the myotonin/protein kinase gene for the diagnosis of the patient. The CTG repeat size and flanking markers of this gene were analyzed for the prenatal diagnosis. Principally, these methods were also used for the analysis the gene abnormality of fragile X syndrome and Huntington disease in which the mechanism of the gene abnormality are similar.2, Analysis of the myotonin/protein kinase gene expression in the lymphoblasts of the disease.We analyzed the myotonin/protein kinase mRNA in lymphoblasts by a PCR technology. A competitive PCR technology was developed to compare the amount of mRNA between the normal and the congenital myotonic dystrophy samples. The expression level of the myotonin/protein kinase gene was not different in the normal and the disease samples. The mRNA amount of the myotonin/protein kinase gene was very low in the lymphoblasts. We will try to use the muscle for this study.3, Expression of the myotonin/protein kinase protein in the E.coli.We tried to make the myotonin/protein kinase protein by a E.coli expression system. The full length myotonin/protein kinase cDNAs were prepared from the human and mouse, and these cDNAs were subcloned into pRSET vectors in the system of E.coli expression. The myotonin/protein kinase protein was not detected in this system clearly. We will try to use the partial fragment of the gene or use other expression system.