TALEN技术在斑马鱼中鉴定MIP新突变位点功能

As the new konckout technology, transcription activator-like effector nucleases (TALENs) is simpler and less toxicity than zinc-finger nucleases (ZFNs).TALENs has been valued and applied widely in the field of genetics, including the genetic eye disorders. A collection of species and genes that have been successfully modified by TALENs, especially in zebrafish. A novol MIP mutation was found in a three generation Chinese progressive congenital cataract family in our recent exome sequencing research. This is the first mutation in the MIP gene has been reported cause progressive congenital cataract in the world. However, this result still needs knockout technology to confirm further. Therefore, we will perform further study of the new valiation in zebrafish using TALE nuclease engineering including targeted genome modification、homologous recombination、gene rescue experiment and whole mount in situ hybridization.We will still perform light microscopy, scanning electron microscopy on the MIP knockout zebrafish to understand the protein well in the lens. In recent years, the studies reveals MIP plays a crucial role in the development of the lens. However, the exactly mechanism of MIP mutation in progressive congenital cataract is still unclear. This mechanism may be more useful in age related cataract research. Therefore, this study is important and necessary.

类转录激活因子效应物核酸酶（TALEN）自2010年底开始成功应用于基因打靶，很快成为一种比锌指核酸酶（ZFN）更容易设计、特异性更高和毒性更低的人工核酸内切酶，是当前用于基因敲除动物制备的最先进的方法，特别是在斑马鱼中的应用。本课题申请人通过外显子组测序发现了基因MIP的一个新的突变可能导致一个三代中国人进展性先天性白内障的发生，这是世界上对该基因导致进展性先天性白内障的首次报道，但目前仍需通过基因敲除进一步验证该位点与先天性白内障的关系。晶状体膜蛋白MIP是含量最丰富的晶状体纤维细胞膜蛋白，对保持晶状体的透明性有十分重要的意义。本课题拟利用TALENs技术，建立MIP基因敲除斑马鱼模型，从而利用光镜、电镜观察基因敲除后晶状体纤维的有序组织结构的变化和晶状体透明性的改变，从而研究该基因导致进展性先天性白内障的原因。

先天性白内障是导致儿童视力障碍和失明的主要原因，遗传因素发挥了重要的作用。本研究的目的是确定常染色体显性遗传先天性进行性点状白内障在中国家庭相关的遗传缺陷并探讨其潜在的发病机制。所有的家庭成员的血液样本收集DNA提取。利用微卫星标记连锁分析。在标记d12s1622得到积极的两点LOD值（ZMAX = 2.71   θ = 0），d12s1724（ZMAX = 2.71  θ = 0），和d12s90（ZMAX = 2.71   θ = 0），所在区域内的主要内在蛋白（MIP）的直接测序揭示了一种新的突变，核苷酸644突变，导致215位密码子发生突变。所有患者均患有先天性进行性点状白内障，但它并没有在健康的成员中出现。生物信息学分析预测，突变的MIP蛋白功能收到影响。我们利用TALEN技术在斑马鱼中将MIP所发现的突变位点后段敲除，发现F2代斑马鱼的体型发生特殊变化，病理切边发现晶体较正常斑马鱼的晶体体积小，可能是由于MIP的突变所引起。