Improved methods for tag and exchange recombineering

标签和交换重组工程的改进方法

• 批准号: 228207-2011
• 负责人: Rancourt, Derrick
• 金额: $ 3.5万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2011
• 资助国家: 加拿大
• 起止时间: 2011-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=478719
• 关键词: Improved; methods; tag; exchange; recombineering

BAC recombineering has revolutionized the generation of gene targeting vectors (TVs). We previously generated over 750 TVs using a combination of high throughput recombineering and Gateway technology. We now propose to develop a TV construction system that would leave the TV sequences seamless. Based upon our experience, there are two key technical problems that interfere with the efficient generation of TVs via tag and exchange recombineering using positive/negative markers. Conventional negative markers do not work effectively for exchange recombineering, as large numbers of false positive BAC clones result, which have undergone illegitimate recombination, deleting the marker without exchange. Second, replacer DNA used to exchange the tag is constructed by PCR, and prone to sequence error, even using high fidelity polymerases.

BAC重组工程已经彻底改变了基因靶向载体（TV）的产生。 我们之前使用高通量重组工程和Gateway技术的组合生成了750多个TV。我们现在建议开发一个电视建设系统，将离开电视序列无缝。 根据我们的经验，有两个关键的技术问题干扰通过使用阳性/阴性标记的标签和交换重组工程有效地产生TV。 常规的阴性标记对于交换重组工程并不有效，因为产生了大量的假阳性BAC克隆，其已经经历了非法重组，在没有交换的情况下删除了标记。 第二，用于交换标签的引物DNA是通过PCR构建的，即使使用高保真聚合酶，也容易出现序列错误。