ANALYSIS OF THE APHAKIA GENE REQUIRED FOR LENS FORMATION

描述先天性或获得性晶状体缺陷，如白内障，是视力受损和失明的主要原因。这项研究提案的长期目标是通过了解晶状体发育和维持的分子机制来改进晶状体疾病的诊断、治疗甚至预防。这项拟议的研究重点是分离和分析小鼠晶状体发育所特需的基因。常染色体隐性无晶状体症(AK)突变纯合子的小鼠由于没有晶状体、虹膜和小眼球缺陷症的瞳孔而失明。在胚胎发育过程中，最早的缺陷是晶状体胎盘有丝分裂上皮细胞纺锤体取向的部分随机化，以及相关细胞外基质(EM)的不规则。将使用位置克隆和位置候选基因相结合的方法来鉴定AK中突变的基因。包含AK基因座的染色体区间将通过利用来自种间杂交的纯合子后代进行多态标记的连锁分析来精细定位。以现有的YAC重叠群为起点，通过构建BAC克隆重叠群进一步细化，然后通过外显子捕获或cDNA选择来鉴定候选基因，从而构建该基因座的高分辨率物理图谱。在适当的发育阶段分离的RNA将被用来评估这些基因在正常眼和猴眼中的表达，并产生用于SSCP和DNA序列分析的突变筛选的cDNA。此外，为了更准确地了解密切连接视杯和晶状体胎盘的EM的组成和作用，将通过免疫定位和原位杂交技术研究已知EM组分在正常和突变眼中的表达。

DESCRIPTION Congenital or acquired defects of the ocular lens such as cataract are a major cause of impaired vision and blindness. The long-term objective of this research proposal is an improved diagnosis, treatment or even prevention of lens disease by means of although understanding of the molecular mechanisms underlying development and maintenance of the lens. The proposed study is focused on isolation and analysis of a gene that is specifically required for lens development in the mouse. Mice homozygous for the autosomal recessive aphakia (ak) mutation are blind due to a absence of lens, iris and pupil with microphtalmia. During embryonic development, the earliest defects a partial randomization of spindle orientation in mitotic epithelial cells of the lens placode, as well as irregularities in the associated extracellular matrix (EM) A combined positional cloning and positional candidate gene approach will be used to identify the gene mutated in ak. The chromosomal interval containing the ak locus will be fine mapped by linkage analysis with polymorphic markers using homozygous offspring derived from an inter-specific cross. A high- resolution physical map of the locus will be constructed using existing YAC contigs as a starting point, further refined by construction of a BAC clone contig, and followed by exon trapping or cDNA selection to identify candidate genes. RNA isolated at appropriate developmental stages will be used to assess the expression of these genes in the normal and monkey eyes, and to generate cDNA to be used in mutation screening by SSCP and DNA sequence analysis. Furthermore,, to gain more precise information about the composition and role of the EM that intimately connects the optic cup with the lens placode, the expression, the expression of known EM constituents will be studied by immunolocalization and in situ hybridization techniques in normal and mutant eyes.