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必要实验工具。一个理想的转基因概括了内源基因的表达模式。为此，它应该包含概括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，因此这种转基因结构通常需要几百kb的DNA长度。大多数动物模型系统基因组的大片段（100- 200kb）已被亚克隆到细菌人工染色体（BACs）中。这些bac可以通过重组（重组基因工程）进行修饰。然而，促进高效BAC转基因的技术是缺乏的。本提案的目标是开发一种技术，有效地插入BAC结构到模式生物斑马鱼的基因组。过去的研究表明，将裸露的BAC DNA或将带有Tol2位点的BAC DNA与Tol2转座酶共同注射到受精的斑马鱼卵中，可获得0.5-5%的转基因率。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BAC转基因方法的通用性，用于含有小片段和大片段DNA的BAC，并作为原理证明，生成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.