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

为了了解人类大脑是如何工作的，以及当它出错时如何进行干预，我们需要能够研究脑细胞，如神经元。为了了解基本的细胞过程在人脑中是如何发生的，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，可用于此类研究。最近，干细胞研究领域发生了一场革命。现在可以从疾病患者身上提取皮肤细胞，将其转化为干细胞，然后在培养中产生患者特定的神经元网络。将新基因插入神经元的能力非常重要，它使科学家能够研究细胞对基因突变等不同触发因素的反应，同时也使他们能够控制细胞的活动。然而，这在细胞如神经元中特别困难，因为它们不分裂，并且通常需要专门的试剂如病毒，其用作载体将基因插入非分裂细胞中。当使用病毒或其他常见的插入方法时，这些基因通常随机插入细胞自身的DNA中，如果插入错误的区域，可能会改变细胞执行正常功能的能力。一种解决方案是修改干细胞的基因组，使其包含一个DNA区域，该区域可以作为安全区域中新基因的“对接点”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成许多不同类型的细胞，因此创建具有基因插入对接位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。在本项目中，我们建议联合收割机结合Axol生物科学的技能，一家专门为所有研究人员提供干细胞衍生神经元的公司，该公司拥有Au神经干细胞组的技能，以创建这样的平台干细胞系。该平台线将通过插入编码视紫红质（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入大脑中观察到的活动类型。这是一个技术上的挑战，将发展双方的技能。Axol的科学家也将在Rhein Parri博士的实验室接受技术培训，这些技术将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，以提高他们的研究能力。这两个交换机在学术和商业环境的经验将建立新的技能在每个组织内，将嵌入在未来的研究和开发，以产生未来的领导者在这一研究领域。

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.