Co-axial Microinjection System for Freezing and Biopsy of Early Embryos

描述(申请人提供)：德古拉吸管是一种同轴胚胎显微注射工具，注射吸管从保持吸管的内部提供。当在它们之间的空间中施加真空时，双嵌套尖端密封在球形胚胎的表面上。这使得胚胎近侧表面的一部分可以在通过玻璃吸管注射、抽吸液体内容物或胚胎细胞活组织检查时被真空保持。在项目开始前不久发明的一种专利激光钻孔活组织检查探头，为植入前胚胎在发育阶段提供了一种新的更好的无害化活组织检查方法，当它们可以为植入前遗传诊断(PGD)提供更准确的DNA信息时。美国专利商标局在申请之日起不到一年的时间内就颁发了“吸管和穿孔”工艺的专利，称没有相关的现有技术可供考虑。该设备本身和我们专有的激光钻孔活组织检查探头的专利正在申请中。2011年10月，该项目被美国国立卫生研究院指定为高度优先项目。这种设备在胚胎发育的所有阶段都有用，直到子宫内植入时为止，它是唯一能够控制和操纵胚胎在孵化胚泡发育阶段的设备。孵化是指从透明带(在最初的几个细胞分裂中包裹卵子和胚胎本身的胶囊)逃脱的自然过程。这些早期胚胎从透明带孵化出来后，没有真正的结构，不能像使用传统设备在仍在透明带的胚胎上所做的那样，从一侧保持并从相反的一侧穿透。德古拉吸管已经证明，它在胚胎冷冻前的水体积减少方面是有价值的。该项目主任泰勒博士使用这种工具，从冷冻/解冻的大羊驼胚胎中培育出了第一批活的婴儿。路易斯安那州立大学的研究人员最近在利用德古拉系统冷冻大型(900微米)马胚胎方面取得了突破性的成功，这在以前是不可能的。蒙大拿州立大学的泰勒博士用小鼠胚胎进行的研究表明，该系统能够从内细胞团中存活地收集几个干细胞，并注入基因 将材料(干细胞)注入小鼠胚胎的胚泡空间以生产转基因小鼠，这一过程以前需要特殊的设备来冷却小鼠的小带，以及昂贵的压电锤来穿透小鼠的小带。人类胚胎可能会作为孵化的囊胚存在两天以上，在植入前的这个分化程度最高的阶段，还没有好的工具来操纵它。因为它很大而且充满液体，所以人类 孵化的囊胚很难成功冷冻，也很难进行活检。虽然Dracula吸管对于其他物种的扩张和孵化的囊胚以及植入前所有阶段的胚胎都将非常重要，但它是唯一有望在8-细胞期之后和植入子宫之前对人类孵化的囊胚进行无害活组织检查的工具。德古拉吸管项目将提供目前最好的设备原型 由几位受人尊敬的胚胎研究人员在各种实验室环境中进行测试和实际使用，主要是在牛和小鼠胚胎模型上工作。顾问将提供关于他们使用该设备的经验的反馈，以及他们为特定目的改进或改变它的想法。发明者和一组技术承包商将致力于纠正发现的设计问题，并将利用适当的微技术公司生产特定的改进原型部件。随着这些评估和升级周期的继续，重点将转移到所有部件的实际制造上，因此，一个坚固、用户友好的设备可以 提交给科学界。这种名为德古拉吸管的工具为常规程序提供了廉价的胚胎操作，如冷冻前的水体积减少，以及对后期胚胎执行以前非常困难或不可能的程序的能力。申请小型企业的GeneSearch公司将通过获得有关知识产权的许可，将德古拉吸管和活组织检查探头商业化。我们将维持一个小型设施，用于创造和测试该设备的新原型和变种，并与被许可方协调，为新用户提供指导和实际援助。

DESCRIPTION (provided by applicant): The Dracula Pipette is a co-axial embryo microinjection tool, with the injection pipette presented from inside the holding pipette. Double nested tips seal against the surface of the spherical embryo when a vacuum is applied in the space between them. This allows a portion of the nearside surface of an embryo to be held by vacuum while a glass pipette is passed for injection, aspiration of fluid contents or biopsy of embryonic cells. A proprietary laser-drilled biopsy probe, invented shortly before the beginning of the project, provides a new and better method for harm-free biopsy of pre-implantation embryos at a stage of development when they can provide more accurate DNA information for pre-implantation genetic diagnosis (PGD). The US Patent and Trademark Office issued the patent for the "suck-and-puncture" process in less than a year from the date of application, saying there was no relevant prior art to consider. Patents for the device itself and for our proprietary laser-drilled biopsy probe are pending. In October of 2011 this project was designated as high priority by NIH. This device, useful at all stages of embryo development up to the time of implantation in the uterus, is uniquely capable of control and manipulation of embryos at the hatched blastocyst stage of development. Hatching refers to the natural process of escape from the zona pellucida (the capsule that encloses the ovum and the embryo itself during the first few cell divisions). After hatching from the zona, these early embryos have no real structure and cannot be held on one side and penetrated from the opposite side, as is done routinely on embryos still in the zona, using conventional equipment. The Dracula Pipette has shown that it is valuable for water-volume reduction before freezing of embryos. Dr. Taylor, the Project Director, produced the first live babies from frozen/thawed embryos of llamas using this tool. Researchers at LSU, have recently had breakthrough success in freezing of large (900um) equine embryos with the Dracula system, which has not been possible before. Work done by Dr. Taylor at Montana State University with mouse embryos shows the capability of this system for survivable collection of a few stem cells from the inner cell mass and for injection of genetic material (stem cells) into the blastocoel space of mouse embryos for production of transgenic mice, a procedure that previously required special equipment for chilling of the zona, as well as the expensive piezo hammer for penetration of the zona. The human embryo may exist for more than two days as a hatched blastocyst, and there has been no good tool for manipulating it at this most-differentiated stage before implantation. Because it is large and fluid-filled, the human hatched blastocyst is difficult to freeze successfully and difficult to biopsy. Though the Dracula Pipette will surely be important for use with the expanded and hatched blastocysts of other species and on embryos at all stages before implantation, it is the only tool on the horizon for harm-free biopsy of human hatched blastocysts beyond the 8-cell stage and before implantation in the uterus. The Dracula Pipette Project will supply the current best prototype of the device for testing and practical use by several respected embryo researchers in a variety of laboratory settings, working mainly with bovine and mouse embryo models. The consultants will provide feedback about their experiences with the device and their ideas for improving or changing it for specific purposes. The inventor and a team of technical contractors will work to correct design problems as they are identified and will use appropriate micro-technology firms for production of specific improved prototype parts. As these cycles of evaluation and upgrade continue, emphasis will shift to practical manufacture of all parts, so a robust, user-friendly device can be presented to the scientific community. This tool, the Dracula Pipette, offers inexpensive embryo manipulation for routine procedures like water- volume reduction before freezing, and the capability to perform previously very difficult or impossible procedures on later-stage embryos. GeneSearch, Inc., the applicant Small Business, will commercialize the Dracula Pipette and biopsy probe through licensing of the intellectual property involved. We will maintain a small facility for creation and testing of new prototypes and variants of the device, and for coordinatin with licensees to provide instruction and practical assistance for new users.