Generation of a site directed gene integration platform for induced pluripotent stem cell lines.

为了了解人脑是如何工作的，以及在出现问题时如何进行干预，我们需要能够研究神经元等脑细胞。要了解人脑中基本的细胞过程是如何发生的，几乎不可能获得活的人类神经元。干细胞提供了产生人类神经元和其他脑细胞的能力，用于此类研究。最近，干细胞研究领域发生了一场革命。现在，从疾病患者身上提取皮肤细胞，将其转化为干细胞，然后在培养中生成患者特有的神经元网络是可能的。将新基因注入神经元的能力非常重要，使科学家能够研究细胞对不同触发因素的反应，如基因突变，但也允许他们控制细胞的活动。然而，这在神经元等细胞中尤其困难，因为它们不会分裂，而且通常需要专门的试剂，如病毒，它们被用作载体，将基因插入未分裂的细胞。当使用病毒或其他常见的插入方法时，这些基因通常被随机插入到细胞自身的DNA中，如果插入到错误的区域，可能会改变细胞执行其正常功能的能力。一种解决方案是修改干细胞的基因组，以加入一个DNA区域，该区域可以作为位于安全区域的新基因的“停靠点”或“发射台”。通过这种方式，新的基因将总是被整合到细胞基因组的同一位置，而不会改变细胞的正常功能。由于干细胞可以分化为许多不同类型的细胞，创造一个带有基因插入对接点的干细胞将为研究人员提供一个平台，以评估插入基因在许多细胞类型中的功能。在这个项目中，我们建议结合Axol Biosciences公司的技术，该公司专门为所有研究人员提供干细胞来源的神经元，并拥有AU神经干细胞小组的技能，以创建这样一个平台干细胞系。这条平台线将通过插入编码通道视紫红质(CHR)蛋白的基因进行测试，这将使神经元对激光敏感，使研究人员能够非侵入性地刺激神经元进行大脑中观察到的活动类型。这是一项技术难度很大的挑战，将发展双方的技能。来自Axol的科学家还将在Rhein Parri博士的实验室内接受技术培训，使他们能够测试他们的细胞系，以确保它们的行为与大脑中的细胞一样。这项工作将为高度专业化的科学家提供这两个领域的独特技能组合，将增强他们的研究能力。学术和商业环境中的交换者的经验将在每个组织中建立新的技能，这些技能将嵌入到未来的研发中，以培养这一研究领域的未来领导者。

To understand how the human brain works and therefore how to intervene when it goes wrong we need to be able to study brain cells such as neurons. Living human neurons are virtually impossible to obtain in order to understand how basic cellular processes occur in the human brain. Stem cells offer the ability to produce human neurons and other brain cells to use in such studies. Recently there has been a revolution in the field of stem cell research. Now it is possible to take skin cells from disease patients, transform them into stem cells, and then from these generate patient specific neuronal networks in culture. The ability to insert novel genes into neurons is of great importance and allows scientists to study cell behaviour in response to different triggers such as gene mutations, but also allows them to control the activity of cells. However this is particularly difficult in cells such as neurons, as they do not divide, and often requires specialised reagents such as viruses, which are used as vectors to insert genes into non-dividing cells. When using viruses or other common insertion methods, these genes are often inserted randomly into the cells own DNA and if inserted in the wrong area, can alter the ability of the cell to carry out its normal functions. One solution is to modify the genome of the stem cell to incorporate a region of DNA that can serve as a 'docking site' or 'launch pad' for novel genes that is in a safe area. In this way novel genes will always be incorporated into the same place in the cells genome without altering the cells normal functions. As a stem cell can be made to differentiate into many different types of cell, creation of a stem cell with a docking site for gene insertion will provide a platform for researchers to assess the function of inserted genes in many cell types.In this project we propose to combine the skills of Axol biosciences, a company who specialises in providing stem cell derived neurons to all researchers with the skills of the Neural stem cell group at AU to create such a platform stem cell line. The platform line will be tested by the insertion of genes encoding Channelrhodopsin (ChR) proteins, which will make the neurons sensitive to laser light, allowing researchers to non-invasively stimulate neurons into the types of activity that are observed in the brain. This is a technically difficult challenge and will develop the skills of both partners. Scientists from Axol will also be trained within Dr Rhein Parri's lab in techniques that will enable them to test their cell lines to ensure that they behave as cells do within the brain. This work will provide highly specialised scientists with the unique combination of skills in both fields that will enhance their research capabilities. The experience of both interchangers in both academic and commercial environments will build new skills within each organisation that will be embedded in future research and development to produce future leaders in this area of research.