Transposon-mediated BAC Transgenesis.

描述(申请人提供)：转基因动物模型是解开正常发育和疾病中基因功能的基本实验工具。理想的转基因重现了内源基因的表达模式。为此，它应该包含重述内源性表达所需的所有调控序列。由于这些调控序列往往相距甚远，这样的转基因构建物通常需要几百千克碱基(Kb)的DNA长度。大多数动物模型系统的基因组大片段(100-200kb)已被亚克隆到细菌人工染色体(BAC)中。这种BAC可以通过重组工程(重组基因工程)进行改造。然而，目前还缺乏促进高效BAC转基因的技术。这项提议的目标是开发一种有效地将BAC构建插入到模式生物斑马鱼基因组中的技术。过去的研究表明，将裸露的BAC DNA注射到斑马鱼受精卵中，或将Tol2转座酶与Tol2位点两侧的BAC DNA共同注射到斑马鱼受精卵中，可产生0.5%-5%的类似转化率。在这里，我们建议测试一种改进的Tol2介导的BAC转基因方法。具体地说，我们打算(1)开发通用工具来将转座酶位点整合到任何BAC中，(2)测试我们改进的Tol2介导的BAC转基因方法的多功能性，用于含有大小DNA片段的BAC，并作为原理的证据，生成Cre报告基因系。

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.