Transposon-mediated BAC Transgenesis.

转座子介导的 BAC 转基因。

• 批准号: 8222984
• 负责人: Holger Knaut
• 金额: $ 8.45万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8222984
• 关键词: Animal Model; Bacterial Artificial Chromosomes; Biological Models; Cells; Chromosomes, Human, Pair 2; Communities; DNA; Development; Disease; Eye; Gene Expression Profile; Gene Transfer Techniques; Genetic; Genetic Engineering; Genome; Genomics; Goals; Injection of therapeutic agent; Length; Libraries; Mediating; Modeling; Nuclear; Recombinants; Reporter; Research; Research Personnel; Site; Staging; Techniques; Testing; Transgenes; Transgenic Animals; Transgenic Organisms; Transposase; Ubiquitin; Vertebral column; Zebrafish; arm; base; egg; gene function; promoter; research study; tool; zebrafish genome

DESCRIPTION (provided by applicant): Transgenic animal models are essential experimental tools for unraveling gene function in normal development and disease. An ideal transgene recapitulates the endogenous gene expression pattern. To this end, it should encompass all the regulatory sequences required for recapitulating endogenous expression. Since these regulatory sequences are often far apart, such transgenic constructs frequently need to be a few hundred kilo bases (kb) of DNA in length. Large fragments (100-200 kb) of the genomes of most animal model systems have been subcloned into bacterial artificial chromosomes (BACs). Such BACs can be modified through recombineering (recombinant genetic engineering). However, techniques that facilitate efficient BAC transgenesis are lacking. The goal of this proposal is to develop a technique for efficient insertion of BAC constructs into the genome of the model organism zebrafish. Past efforts show that injection of naked BAC DNA or co-injection of Tol2 transposase with BAC DNA flanked by Tol2 sites into fertilized zebrafish eggs results in comparable transgenesis rates of 0.5-5%. Here, we propose to test a modified Tol2-mediated BAC transgenesis approach. Specifically, we intend to (1) develop universal tools for incorporating transposase sites into any BAC, (2) test the versatility of our modified Tol2-mediated BAC transgenesis approach for use with BACs harboring small and large DNA fragments and, as a proof of principle, generate Cre reporter lines. PUBLIC HEALTH RELEVANCE: Transgenic zebrafish models are essential for our understanding of gene function in normal development and disease. While transgenesis of small DNA constructs is a standard technique in zebrafish, the integration of 100 kilo bases or more into the zebrafish genome is hampered by its low efficiency of transgenesis. In this proposal, we intend to develop a technique for efficient integration of large transgenes into the zebrafish genome.

描述（由申请人提供）：转基因动物模型是揭示正常发育和疾病中基因功能的重要实验工具。理想的转基因概括了内源基因的表达模式。为此，它应该涵盖重演内源表达所需的所有调控序列。由于这些调控序列通常相距很远，因此此类转基因构建体的 DNA 长度通常需要数百个碱基 (kb)。大多数动物模型系统的基因组大片段（100-200 kb）已被亚克隆到细菌人工染色体（BAC）中。此类 BAC 可以通过重组工程（重组基因工程）进行修改。然而，缺乏促进有效 BAC 转基因的技术。该提案的目标是开发一种将 BAC 构建体有效插入模式生物斑马鱼基因组的技术。过去的研究表明，将裸露的 BAC DNA 注射或将 Tol2 转座酶与侧翼为 Tol2 位点的 BAC DNA 共同注射到受精斑马鱼卵中，可产生 0.5-5% 的可比转基因率。在这里，我们建议测试一种改良的 Tol2 介导的 BAC 转基因方法。具体来说，我们打算 (1) 开发用于将转座酶位点整合到任何 BAC 中的通用工具，(2) 测试我们改良的 Tol2 介导的 BAC 转基因方法的多功能性，以用于包含小和大 DNA 片段的 BAC，并作为原理证明，生成 Cre 报告基因系。 公共卫生相关性：转基因斑马鱼模型对于我们了解正常发育和疾病中的基因功能至关重要。虽然小 DNA 构建体的转基因是斑马鱼的标准技术，但将 100 公斤或更多碱基整合到斑马鱼基因组中却因其转基因效率低而受到阻碍。在本提案中，我们打算开发一种将大型转基因有效整合到斑马鱼基因组中的技术。