ANALYSIS OF THE APHAKIA GENE REQUIRED FOR LENS FORMATION

先天性或后天的晶状体缺陷，如白内障，是视力受损和失明的主要原因。本研究计划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善晶状体疾病的诊断、治疗甚至预防。这项研究的重点是分离和分析小鼠晶状体发育特别需要的基因。纯合常染色体隐性无晶状体（ak）突变的小鼠由于晶状体、虹膜和瞳孔的缺失而失明。在胚胎发育过程中，最早的缺陷是晶状体位有丝分裂上皮细胞的纺锤体取向的部分随机化，以及相关的细胞外基质（EM）的不规则性。一种结合定位克隆和定位候选基因的方法将用于鉴定ak中的突变基因。利用来自种间杂交的纯合后代，利用多态性标记进行连锁分析，可以精确定位含有ak位点的染色体间隔。该基因座的高分辨率物理图谱将以现有的YAC基因组为起点，通过构建BAC克隆基因组进一步完善，然后通过外显子捕获或cDNA选择来鉴定候选基因。在适当发育阶段分离的RNA将用于评估这些基因在正常和猴子眼睛中的表达，并生成cDNA用于SSCP和DNA序列分析的突变筛选。此外，为了获得关于密切连接视杯和晶状体基板的EM的组成和作用的更精确信息，将通过免疫定位和原位杂交技术研究正常和突变眼睛中已知EM成分的表达。

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.