Transposon-mediated BAC Transgenesis.

转座子介导的 BAC 转基因。

• 批准号: 8442282
• 负责人: Holger Knaut
• 金额: $ 8.02万
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8442282
• 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.

描述（由申请人提供）：转基因动物模型是在正常发育和疾病中阐明基因功能的必要实验工具。理想的转基因概括了内源基因表达模式。为此，它应涵盖概括内源性表达所需的所有调节序列。由于这些调节序列通常相距较远，因此这种转基因构建体经常需要数百公斤（kb）的DNA长度。大多数动物模型系统的基因组的大片段（100-200 kb）已被亚克隆成细菌人造染色体（BAC）。可以通过重组（重组基因工程）来修改此类BAC。但是，缺乏促进有效BAC转基因的技术。该提案的目的是开发一种技术，以有效地将BAC构建体插入模型生物体斑马鱼的基因组中。过去的努力表明，注射裸露的BAC DNA或将TOL2转座酶与BAC DNA共同注射，而BAC DNA侧面是TOL2位点，导致受精卵的斑马鱼卵，导致可比的转基因率为0.5-5％。在这里，我们建议测试一种修饰的TOL2介导的BAC转基因方法。具体而言，我们打算（1）开发用于将转座酶位点掺入任何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，用于与具有大小的大型DNA片段的BAC一起使用，并作为原理证明，生成CRE报告。