Creation of Alzheimer's disease model neuron by embryonic and genetic engineering

我们这个项目的最终目标是创造出携带阿尔茨海默病（AD）异常的活神经元，以研究AD并开发治愈性疗法。基本的策略是，我们构建了小鼠胚胎干细胞（ES细胞），其中AD致病性V642 I突变引入内源性淀粉样前体蛋白（APP）基因，结合一种方案，以有效地分化ES细胞为有丝分裂后神经元，而不使用细胞分选仪。通过这种方案，ES细胞分化为超过90%的成年有丝分裂后神经元的中枢型。V642 I-APP基因敲入的ES细胞来源的神经元表现出显著增加的Abeta 42分泌。我们用基因芯片分析了这些突变kock-in神经元的基因表达，并用真实的时间PCR进行了验证。到目前为止，与野生型ES细胞相比，V642 I-APP和I213 T-PS1 kock-in ES细胞来源的神经元中有7个基因的表达增加。我们正在努力制备G93 R-SOD 1 kock-in或转基因ES细胞，以比较这些神经退行性模型神经元。我们正在寻找最有效的从ES细胞分化为运动神经元的方法。目前，我们正在将ES细胞衍生的神经元与产生音刺猬的细胞共培养以分化成运动神经元。

Our final goal of this project is to create live neurons harboring Alzheimer' s disease(AD) abnormalities to study AD and to develop curative therapies. Basic strategy is that we constructed mouse embryonic stem(ES) cells, in which the AD-causative V642I mutation was introduced to the endogenous amyloid precursor protein(APP) gene, in combination with a protocol to efficiently differentiate ES cells into postmitotic neurons without using a cell sorter.By this protocol, ES cells differentiated into more than 90% of the central type of adult postmitotic neurons.. The neurons derived from V642I-APP knock-in ES cells exhibited significantly increased Abeta 42 secretion.Next, we created neurons derived from I213T-PS1 knock-in ES cell with the similar protocol. We analyzed gene expression of these mutation kock-in neurons using microarray analysis and validated with real time PCR. So far, expression of seven genes from neurons derived from V642I-APP and I213T-PS1 kock-in ES cells was increased compared with the one from wild type ES cells.In addition, we are trying to create other neurodegenerative disease model neurons. We are working to prepare either G93R-SOD1 kock-in or transgenic ES cells to compare these neurodegenerative model neurons.. We are searching most effective differentiating protocol from the ES cells to motoneurons. Currently, we are co-culturing the ES cell derived neurons with sonic hedgehog producing cells to differentiate into motoneurons.