IDBR: Using a Nanopore to Transfect Cells with Single Molecule Precision to Induce Pluripotency Efficiently in Fibroblasts

IDBR：使用纳米孔以单分子精度转染细胞，有效诱导成纤维细胞的多能性

• 批准号: 1256052
• 负责人: Gregory Timp
• 金额: $ 55.06万
• 依托单位: University of Notre Dame
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1256052&HistoricalAwards=false
• 关键词: IDBR; Using; Nanopore; Transfect; Cells

ABSTRACTAn award is made to the University of Notre Dame to develop a new tool that uses a synthetic nanometer-diameter pore (i.e. a nanopore) to reprogram the genetic memory of specialized cells, inducing them to become like embryonic stem cells. Stem cell research promises regenerative therapies for everything from blindness, to the repair of heart and nerve damage, and even aging. Until recently, clinical research has focused mainly on two kinds of cells: embryonic and non-embryonic or adult stem cells. Beyond the ethical dilemma posed by the use of embryonic stem cells, both cell types are scarce relative to the level required for efficacious treatment. On the other hand, induced pluriopotent stem cells (iPSCs) may now represent a viable alternative. iPSCs are derived from specialized somatic cells by forcing the expression of four key genes; they are similar to embryonic stem cells. Unfortunately, only about 0.01-0.1% of human somatic cells can be forced to become iPSCs. The reprogramming efficiency of somatic cells is crippled by the lack of control over the introduction and level of expression of the key genes. This research effort at Notre Dame will produce a tool that uses a nanopore to transfer nucleic acids into a single cell one molecule at a time, affording stringent control over the expression of the target genes. The synthetic nanopore uses a tightly confined electric field to create a pore in the cell membrane that facilitates the delivery of nucleic acids and even whole genes potentially with single molecule precision, while allowing for serial or repeated gene delivery into the same cell. As a crucible for testing the tool, nucleic acids encoding the key genes will be introduced into somatic cells to produce iPSCs.This research has broader implications beyond the development of a tool for gene delivery into cells. This project also presents an opportunity to understand and transform biology through control of the gene and its environment with single molecule precision. To take advantage of this opportunity, the research will be translated into educational curricula and disseminated into the biological, health and medical communities. There are several components to this outreach program: i. teaching one-another through a monthly seminar series and a web-site; ii. inspiring students through novel curricula promulgated through a new lab course, Introduction to Systems Biology, taught under the Engineering and Biological Sciences rubrics; and finally, iii enlightening the public and high school students, in particular, about the impact of this novel nanotechnology on biology and medicine through partnerships with a local New Tech High School.

摘要美国圣母大学获得一项奖项，以开发一种新的工具，该工具使用合成的纳米直径孔（即纳米孔）来重新编程特化细胞的遗传记忆，诱导它们变得像胚胎干细胞一样。干细胞研究有望为从失明到修复心脏和神经损伤，甚至衰老的一切提供再生疗法。直到最近，临床研究主要集中在两种细胞上：胚胎干细胞和非胚胎干细胞或成体干细胞。除了使用胚胎干细胞所带来的伦理困境之外，这两种细胞类型相对于有效治疗所需的水平都很稀缺。另一方面，诱导多能干细胞（iPSC）现在可能是一种可行的替代方案。iPSC是通过强制表达四个关键基因而从特化的体细胞中衍生出来的;它们类似于胚胎干细胞。不幸的是，只有大约0.01-0.1%的人类体细胞可以被迫成为iPSC。由于缺乏对关键基因的导入和表达水平的控制，体细胞的重编程效率受到削弱。Notre Dame的这项研究工作将产生一种工具，该工具使用纳米孔将核酸一次一个分子转移到单个细胞中，从而对靶基因的表达进行严格控制。合成纳米孔使用紧密限制的电场在细胞膜中产生孔，其促进核酸甚至整个基因的递送，可能具有单分子精度，同时允许连续或重复的基因递送到同一细胞中。作为测试该工具的坩埚，编码关键基因的核酸将被引入体细胞中以产生iPSCs。这项研究的意义不仅限于开发基因递送到细胞中的工具。该项目还提供了一个通过单分子精确控制基因及其环境来理解和改造生物学的机会。为了利用这一机会，将把这项研究转化为教育课程，并向生物、卫生和医学界传播。这个推广计划有几个组成部分：一。通过每月一次的系列研讨会和一个网站相互教授;二.通过一个新的实验室课程，系统生物学导论，根据工程和生物科学的标题下教授颁布的新课程激励学生;最后，iii启发公众和高中学生，特别是通过与当地新技术高中的合作伙伴关系，这种新的纳米技术对生物学和医学的影响。