Using site-specific nucleases to manipulate the zebrafish genome

使用位点特异性核酸酶操纵斑马鱼基因组

• 批准号: 8896018
• 负责人: NATHAN D LAWSON
• 金额: $ 41.25万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8896018
• 关键词: Address; Adult; Alleles; Animal Model; Animals; Applications Grants; Architecture; Area; Award; Behavior; Binding; Biological; Biology; Blood Cells; Blood Vessels; Cells; Characteristics; Chromatin Loop; Code; Communities; Coupled; DNA; DNA Binding; DNA Binding Domain; Defect; Development; Developmental Biology; Disease; Disease model; Embryo; Embryonic Development; Endothelial Cells; Engineering; Foundations; Frequencies; Gene Silencing; Gene Targeting; Generations; Genes; Genetic Determinism; Genetic Screening; Genome; Genomic Segment; Genomics; Globin; Goals; Grant; Health; Hematopoiesis; Imagery; Investigation; Knock-out; Length; Lesion; Link; Locus Control Region; Methodology; Methods; MicroRNAs; Modeling; Modification; Morphogenesis; Mutagenesis; Mutation; Neurofibromatoses; Physiology; Process; Proteins; Protocols documentation; Reagent; Regulatory Element; Research; Research Personnel; Resources; Role; Site; Somatic Cell; Study models; Technology; Testing; Tissues; Transcriptional Regulation; Untranslated RNA; Work; Zebrafish; Zinc Fingers; base; blood vessel development; cell type; design; functional improvement; gene function; gene replacement; genetic approach; genome editing; human disease; improved; insertion/deletion mutation; insight; interest; knockout gene; mutant; new technology; novel; nuclease; preference; repaired; reverse genetics; tool; transmission process; zebrafish development; zebrafish genome

DESCRIPTION (provided by applicant): The zebrafish has characteristics that make it an ideal model organism for studying genetic determinants that participate in development and disease. The advent of zinc finger nucleases, and more recently TAL-effector nucleases, has provided an accessible methodology for the targeted disruption of practically any gene within the zebrafish genome. However limitations still remain to the application of these site-specific nucleases (SSNs) in zebrafish, in particular for the generation of large deletions or tailor-made modifications to large coding (or non-coding) regions of the genome. Realizing this goal is critical to exploiting the full potential of the zebrafish as a developmental and disease model. In this grant application, we will rely on our established expertise in the field to optimize and exted the use of SSNs in zebrafish. To provide a foundation for our genome editing efforts, initial studies in Aim 1 will focus on increasing the efficiency of double-strand break formation by SSNs through improvements in the nuclease architecture. These improvements will be coupled to new methods to optimize the ratio of germline to somatic lesion frequency. In parallel, we will test the application of improved SSNs to rapidly interrogate gene function through efficient targeted biallelic somatic cell knockout. In particular, we will focus on development of approaches to allow biallelic gene knockout in a restricted somatic cell-type to facilitate analysi of cell autonomy. In Aim 2, we will apply SSNs to expand the repertoire of desired lesions that can be introduced at targeted sites in the zebrafish genome. This will include the application of SSNs to introduce large deletions and inversions through the use of multiple SSN pairs, as well as incorporation of domains that facilitate chromatin looping to generate efficient deletion of intervening genomic segments. We will also apply a similar approach to replace deleted regions with an exogenously supplied donor DNA to allow tailor-made alteration of the zebrafish genome. In Aim 3, we will apply improved SSNs to determine the function of non-coding sequences in the zebrafish genome during hematopoiesis and vascular development. In particular, we will introduce targeted deletions in the locus control (LCR) region of the major globin locus to determine its importance for globin switching during embryonic development. In parallel, we will apply SSNs to generate targeted deletions in miR-126a and b to determine the distinct roles of these microRNAs during flow- dependent and -independent vascular morphogenesis. The advances made in the context of the studies proposed in this application will enable the zebrafish community to create a variety of tailored genomic manipulations to facilitate detailed investigation of gene function. As we have in the past, we will continue to share all protocols and reagents that are developed in the course of these studies to facilitate their application within the community.

描述(申请人提供)：斑马鱼的特征使其成为研究参与发育和疾病的遗传决定因素的理想模式生物。锌指核酸酶和最近的TAL效应核酸酶的出现，为斑马鱼基因组中几乎任何基因的靶向干扰提供了一种可行的方法。然而，这些位点特异性核酸酶(SSN)在斑马鱼中的应用仍然存在局限性，特别是在产生大的缺失或对基因组的大编码(或非编码)区域进行量身定制的修改方面。实现这一目标对于充分发挥斑马鱼作为发育和疾病模型的潜力至关重要。在……里面 在本次资助申请中，我们将依靠我们在该领域的成熟专业知识来优化和扩大SSN在斑马鱼中的使用。为了为我们的基因组编辑工作提供基础，目标1的初步研究将集中在通过改进核酸酶结构来提高SSNS形成双链断裂的效率。这些改进将与优化种系与体细胞损伤频率比率的新方法相结合。同时，我们将测试改进的SSNS的应用，通过高效的靶向双等位基因体细胞敲除来快速询问基因功能。特别是，我们将专注于开发允许在受限体细胞类型中进行双等位基因敲除的方法，以促进对细胞自主性的分析。在目标2中，我们将应用SSN来扩展可在斑马鱼基因组中的目标位置引入的所需损伤的谱系。这将包括应用SSN通过使用多个SSN对来引入大的缺失和倒置，以及加入促进染色质环路的结构域，以产生有效的插入基因组片段的删除。我们还将应用类似的方法，用外源提供的供体DNA取代缺失的区域，以便对斑马鱼基因组进行量身定做的改变。在目标3中，我们将应用改进的SSNS来确定斑马鱼基因组中非编码序列在造血和血管发育过程中的功能。特别是，我们将在主要珠蛋白基因座的位置控制区(LCR)引入有针对性的缺失，以确定其在胚胎发育过程中对珠蛋白转换的重要性。同时，我们将应用SSNS在miR-126a和b中产生靶向缺失，以确定这些microRNAs在流依赖和非依赖血管形态发生过程中的不同作用。在本申请中提出的研究背景下取得的进展将使斑马鱼界能够创建各种量身定做的基因组操作，以促进对基因功能的详细研究。一如过往，我们会继续分享在这些研究过程中制订的所有方案和试剂，以促进它们在社会上的应用。