Single Nucleotide Genome Modifications in Oocytes

描述（由申请人提供）：我们结合了几种新技术，并将其应用于卵母细胞和胚胎，目标是快速，高效，有效地改变动物的基因组。我们利用卵母细胞和早期胚胎在非转基因的方法来改变特定的基因功能。卵母细胞将用于F0代构建稳定的转基因系，胚胎用于纯合基因改变以快速筛选基因。这项技术将为新生物体的研究打开大门，既可以修改基因进行功能分析，也可以纠正被认为是亚功能的基因。由于能够引入外源DNA结构，基因组操作方法主要是在培养细胞和遗传上可处理的胚胎中进行的。然而，为了创造稳定的转基因动物，这些方法需要反向杂交，以减少基因嵌合或整合到种系中，从而增加了时间和资源的成本。这些限制极大地降低了在生物研究的关键生物体中广泛利用的有效性，特别是那些遗传可追溯性低的生物体。利用转录激活因子样效应（TALE）功能连接DNA脱氨酶活性的酶结构域进入卵母细胞是一种新颖的方法，该项目的成功将使研究人员能够在F0代构建基因改变。本项目利用ADAR的催化结构域，在预定位点制作单核苷酸基因组靶向系统。我们最近的初步结果证明了该结构域的dna脱氨活性，加上一个可证明的tale靶向机制，增加了该项目的可行性。这个项目的结果将对所有的研究人员有用，特别是那些在遗传上不容易处理的生物中工作的人，以及那些希望在没有转基因方法的情况下快速筛选基因功能的人。我们相信这项技术将从基因研究的白话中去除非模式生物的措辞，并且在未来可能通过纠正与先天性疾病状态相关的点突变而适用于临床应用。

DESCRIPTION (provided by applicant): We combine several new technologies and apply them to oocytes and embryos with the goal of quickly, efficiently, and effectively altering genomes of animals. We utilize oocytes and early embryos in a non-transgenic approach to alter specific gene function. Oocytes will be used for construction of stable transgenic lines at the F0 generation, and embryos for homozygous gene alteration for quick gene screening. This technology will open the door for the study of new organisms and to either modify a gene for functional analysis or correct a gene that is otherwise rendered sub-functional. Genome manipulation approaches have been performed mostly with cultured cells and genetically tractable embryos because of the capabilities to introduce exogenous DNA constructs. To create stable transgenic animals, however, these approaches require back-crosses to reduce genetic mosaicism or integration into the germ line and thus increase the cost of time and resources. These limitations minimize the effectiveness for broad utilization in organisms key for biological research, especially those whose genetic tractability is low. The use transcriptional activator like effector (TALE) functions linked to an enzymatic domain of DNA deaminase activity into oocytes is novel, and success of this project will allow researchers to construct gen alterations at the F0 generation. This project crafts a single nucleotide genome targeting system at a predetermined site by use of the catalytic domains of ADAR. Our recent preliminary results demonstrate DNA-deaminating activity of this domain and coupled with a demonstrable TALE-targeting mechanism increases feasibility of this project. The outcome of this project will be useful to all researchers, especially those working in genetically less-tractable organisms, and those wishing to rapidly screen gene functions without transgenic approaches. We believe this technology will remove the wording non-model organism from the genetic research vernacular, and in the future may be applicable for clinical use by correcting a point mutation related to a congenital disease state.