Analysis of Cytoplasmic Prion Protein Toxicity

描述(申请人提供)：上世纪90年代中期S提供的证据表明，牛海绵状脑病(俗称疯牛病)很可能是由人传播给人类的。人们普遍认为导致这些疾病的病原体是变形异常的普恩蛋白。除了感染途径，人类还可以零星地或通过表达突变的普恩蛋白(外显率约为100%)而患上普恩疾病。最近的工作指出，胞质是Pron蛋白的毒性部位。突变的Prion蛋白可能有很高的毒性，因为它们错误折叠的可能性增加，因此错误定位到细胞质中。为了研究突变的Prion蛋白的毒性增加的机制及其与胞浆Pron毒性的关系，将对ES细胞进行基因改造，将突变的Prion蛋白直接表达到胞浆中。这些细胞系将被分化为神经元、GILA或成纤维细胞，并用于研究这些突变对PrP的亚细胞运输、降解和毒性的影响。基因打靶的方法将允许这些构建物在mRNA水平上同等表达的可能性很高。选择ES细胞作为模型系统将使细胞培养实验有效地过渡到小鼠身上。

DESCRIPTION (provided by applicant): Evidence produced in the mid 1990's suggests that bovine spongiform encephalopathy (commonly known as mad cow disease) was probably transmitted to humans. The agents widely believed to be responsible for these diseases are abnormally shapened prion proteins. Along with an infectious route, humans can also develop prion diseases either sporadically or by expressing a mutant prion protein (penetrance approximately 100%). Recent work points to the cytosol as being the site of toxicity of prion proteins. Mutant prion proteins may be highly toxic because they have an increased probability of misfolding and therefore mislocalizing to the cytosol. To study the mechanism of increased toxicity of the mutant prion proteins and how it relates to cytosolic prion toxicity, ES cells will be genetically altered to express mutant prion proteins directly into the cytosol. These cell lines will be differentiated into neurons, gila, or fibroblasts and used to study the effects these mutations have on subcellular traffciking, degradation, and toxicity of PrP. The gene-targeting approach will allow for a high probability of equivalent expression of these constructs at the mRNA level. The selection of ES cells as a model system will permit efficient transition of cell culture experiments into mice.