Transposon-mediated BAC Transgenesis.

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表达模式。为此，它应该包括重演内源性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通常需要几百千碱基（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）已被亚克隆到细菌人工染色体（BAC）中。这样的BAC可以通过重组工程（重组基因工程）来修饰。然而，缺乏促进有效BAC转基因的技术。本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。过去的努力表明，将裸BAC DNA注射或将Tol 2转座酶与侧翼为Tol 2位点的BAC DNA共注射到受精的斑马鱼卵中导致0.5- 5%的可比转基因率。在这里，我们建议测试一种改良的Tol 2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并入任何BAC的通用工具，（2）测试我们的经修饰的Tol 2介导的BAC转基因方法用于携带小和大DNA片段的BAC的多功能性，并且作为原理证明，产生Cre报告细胞系。

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.