Single Nucleotide Genome Modifications in Oocytes

卵母细胞中的单核苷酸基因组修饰

• 批准号: 8691207
• 负责人: GARY M WESSEL
• 金额: $ 20.31万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-31 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8691207
• 关键词: Animals; Back; Base Pairing; Basic Science; Biological Sciences; Biomedical Research; Birth Rate; Catalytic Domain; Cell physiology; Cleaved cell; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Coupled; Cultured Cells; DNA; DNA Sequence; DNA-Binding Proteins; Deaminase; Devices; Disease; Effectiveness; Embryo; Embryonic Development; Engineering; Enzymes; Epilepsy; Essential Amino Acids; Fertilization; Fetal Development; Future; Gene Mutation; Gene Transfer Techniques; Generations; Genes; Genetic; Genetic Research; Genetically Modified Animals; Genome; Genomics; Germ Lines; Goals; Hereditary Disease; Human; Link; Live Birth; Longevity; Medical; Messenger RNA; Methodology; Modification; Monitor; Mosaicism; Mus; Mutate; Mutation; Nucleotides; Oocytes; Organism; Outcome; Point Mutation; Positioning Attribute; Production; Protein Engineering; Proteins; Quality of life; Reporter; Research; Research Personnel; Resources; Risk; Site; Speed; Starfish; System; Technology; Terminator Codon; Testing; Time; Transcription Coactivator; Transgenic Animals; Transgenic Organisms; Translational Research; Work; base; biological research; cost; design; ds-DNA; egg; gene function; gene replacement; genetic manipulation; mutant; nervous system disorder; new technology; novel; novel strategies; public health relevance; screening; stem; success; tool

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.

描述（由申请人提供）：我们结合了联合收割机几种新技术，并将其应用于卵母细胞和胚胎，目的是快速，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的非转基因方法来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，胚胎用于纯合基因改变，以进行快速基因筛选。这项技术将为研究新的生物体打开大门，并修改基因进行功能分析或纠正基因，否则将呈现亚功能。基因组操作的方法已经进行了大部分与培养的细胞和遗传易处理的胚胎，因为引入外源DNA构建体的能力。然而，为了创造稳定的转基因动物，这些方法需要回交以减少遗传嵌合或整合到种系中，从而增加时间和资源的成本。这些限制使生物研究的关键生物体，特别是那些遗传易处理性低的生物体的广泛利用的有效性最小化。使用与DNA脱氨酶活性的酶结构域相关的转录激活因子样效应子（TALE）功能进入卵母细胞是新颖的，该项目的成功将使研究人员能够在F0代构建基因改变。本项目利用阿达尔的催化结构域在预定位点构建单核苷酸基因组靶向系统。我们最近的初步结果表明，该结构域的DNA脱氨活性，加上一个可证明的TALE靶向机制，增加了这个项目的可行性。这个项目的结果将是有用的所有研究人员，特别是那些在遗传学上不太听话的生物工作，和那些希望快速筛选基因功能，而不转基因的方法。我们相信这项技术将从遗传研究术语中删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使用。