Single Nucleotide Genome Modifications in Oocytes

描述(申请人提供)：我们结合了几项新技术，并将其应用于卵母细胞和胚胎，目标是快速、高效和有效地改变动物的基因组。我们利用卵母细胞和早期胚胎以非转基因的方式改变特定的基因功能。卵母细胞将用于构建F0代稳定的转基因品系，胚胎用于纯合子基因改变，用于快速基因筛选。这项技术将为研究新的生物体打开大门，或者修改用于功能分析的基因，或者纠正否则被认为是亚功能的基因。基因组操作方法主要是对培养细胞和遗传上易处理的胚胎进行的，因为它们具有引入外源DNA结构的能力。然而，为了创造稳定的转基因动物，这些方法需要回交来减少遗传嵌合体或整合到生殖系中，从而增加时间和资源的成本。这些限制降低了广泛应用于生物研究的关键生物体的有效性，特别是那些基因易驯化程度低的生物体。将转录激活因子样效应器(TALE)功能与DNA脱氨酶活性的酶结构域连接到卵母细胞中是新的，该项目的成功将使研究人员能够构建F0代的基因改变。这个项目利用ADAR的催化域，在预定的位置制作了一个单核苷酸基因组靶向系统。我们最近的初步结果证明了这个结构域的DNA脱氨活性，再加上一个可证明的故事靶向机制，增加了这个项目的可行性。该项目的结果将对所有研究人员有用，特别是那些在遗传不太容易控制的生物中工作的研究人员，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删除非模式生物的措辞，并在未来通过纠正与先天性疾病状态有关的点突变而应用于临床。

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.