Transfection of Lens Cells with Phakinin Mutants

描述（由申请方提供）：拟定研究的长期目标是确定晶状体特异性珠丝蛋白（BFP）细胞骨架如何有助于晶状体清晰度。我们的假设是，最近发现的突变BF亚基phakinin与人类白内障形成中断BF自组装或生化处理。具体目标1将检验以下假设：在体外条件下或在用融合至标记蛋白标签的BF亚基构建体转染培养细胞期间，BFP的原位生物化学特征未改变。这将在培养的鸡透镜环形垫细胞中用共聚焦显微镜和免疫化学检测来完成，所述细胞用融合至荧光蛋白（FP）或HA表位的BF亚基构建体转染。具体目标2将检验以下假设：与人白内障相关的phakinin突变干扰体外透镜纤维分化期间BFP的聚合和/或生化加工。这将在用与荧光蛋白或抗原决定簇融合的突变的噬菌体激肽亚基转染的培养细胞中实现。BF聚合和生化处理将通过共聚焦显微镜和表达蛋白的免疫化学检测进行监测。这些研究的结果将导致更好地了解珠状细丝的功能和某些形式的白内障的病因。

DESCRIPTION (provided by applicant): The long-term objectives of the proposed studies are to determine how the lens-specific beaded filament protein (BFP) cytoskeleton contributes to lenticular clarity. Our overlying hypothesis is that recently identified mutations in the BF subunit phakinin associated with human cataract formation interrupt BF self-assembly or biochemical processing. Specific Aim 1 will test the hypothesis that the in situ biochemical characteristics of BFPs are not altered under in vitro conditions or during the transfection of cultured cells with BF subunit constructs fused to marker protein tags. This will be accomplished with confocal microscopy and immunochemical detection in cultured chicken lens annular pad cells transfected with BF subunit constructs fused to fluorescent proteins (FPs) or the HA epitope. Specific Aim 2 will test the hypothesis that phakinin mutations associated with human cataracts interfere with the polymerization and/or biochemical processing of BFPs during in vitro lens fiber differentiation. This will be accomplished in cultured cells transfected with mutated phakinin subunits fused to fluorescent proteins or antigenic determinants. BF polymerization and biochemical processing will be monitored with confocal microscopy and immunochemical detection of the expressed proteins. The results of these studies will lead to a better understanding of beaded filament function and the etiology of some forms of cataract.