Systematic Identification of Tunable Transfection Reagents for Stem Cell Biology

人胚胎干细胞（hES）具有巨大的研究兴趣，并且由于其多能性或发育成大量分化细胞类型之一的能力而提供了治疗价值的希望。然而，目前对ES细胞的研究受到基于非病毒脂质介导的DNA递送的标准技术所观察到的低转化效率的阻碍。转化是将DNA引入细胞的能力，并且允许特定蛋白质的过表达、“敲低”或生物化学“标记”，并且是评估给定蛋白质对细胞的贡献的标准手段。我们建议通过建立一个系统的和结果知情的搜索新的和有效的试剂，以化学方式协助干细胞的转染，以克服这一技术障碍。这将通过结合两个研究小组的专业知识来进行，剑桥（Florian Hollfelder）在化学合成、文库开发和高通量筛选方面拥有丰富的经验，而伯明翰（Karl Nightingale和Laura奥尼尔）的研究计划集中在使用原代和已建立的白血病细胞系以及小鼠和人类ES细胞。具体而言，我们建议产生广泛的和化学多样的改性聚乙烯亚胺（PEI），广泛用于细胞转染的试剂库，并建立高通量筛选，以确定有效的和无毒的试剂。如果需要，将对有效试剂进行结构/活性分析，以告知二级文库合成。该文库将针对许多技术难度越来越大的组织培养细胞类型进行筛选。最初，我们建议使用称为HL 60细胞的白血病细胞系。这代表了一种理想的细胞系，因为它生长迅速，容易进行筛选，但也是一种难以筛选的细胞类型-这应该有助于建立干细胞的筛选条件。重要的是，由于HL 60细胞广泛用于研究实验室，是急性髓性白血病的良好模型，因此任何鉴定的试剂都将立即引起普遍关注。然后，我们建议利用从这个初始阶段获得的经验，针对几种小鼠ES细胞，并最终针对几种人类ES细胞重新筛选文库。该提案的一个重要部分是，经鉴定的试剂将接受一系列“质量控制”检查。这将主要是在功能水平上（或确保细胞继续像干细胞一样发挥作用），但我们也将检查试剂是否可以执行分子生物学家希望使用它们进行的各种实验。

Human embryonic stem (hES) cells are of great research interest, and offer the promise of therapeutic value, due to their pluripotency, or ability to develop into one of a large number of differentiated cell types. However, research on ES cells is currently hampered by the low transformation efficiencies observed by standard technologies based on non-viral lipid-mediated DNA delivery. Transformation is the ability to introduce DNA into cells, and permits the over-expression, 'knock-down' or biochemical 'tagging' of a specific protein, and is a standard means to assess the contribution of a given protein to the cell. We propose to overcome this technical barrier by establishing a systematic and results-informed search for new and effective reagents to chemically assist the transfection of stem cells. This will be performed by combining the expertise of two research groups, Cambridge (Florian Hollfelder) has broad experience of chemical synthesis, library development and high throughput screening, whereas Birmingham ( Karl Nightingale & Laura O'Neill) have research programmes centred on the use of primary and established leukaemic cell lines together with mouse and human ES cells. Specifically, we propose to generate a broad and chemically diverse library of modified polyethylene imines (PEIs), a reagent widely used in cell transfection, and establish a high throughput screen to identify effective and non-toxic reagents. Effective reagents will be subjected to structure/activity analysis to inform secondary library synthesis if necessary. This library will be screened against a number of tissue culture cell types of increasing technical difficulty. Initially we propose to use a leukaemic cell line called HL60 cells. This represents an ideal cell line for troubleshooting the screen as it grows quickly and easily, but is also a difficult cell type to transfect - this should be useful in establishing screen conditions for the stem cells. Importantly, any reagents identified will of immediate general interest as HL60 cells are widely used in research labs, being a good model of acute myeloid leukaemia. We then propose to utilise the experience gained from this initial phase to re-screen the library against several sorts of mouse ES cells, and finally against several sorts of human ES cells. An important part of the proposal at that the identified reagents will be subjected to a number of 'quality control' checks. This will primarily be at a functional level (or ensuring that the cells continue acting like stem cells), but we will also check that the reagents can perform the sorts of experiments that molecular biologists would want to use them for.