Regeneron Pharmaceuticals, Inc, Tarrytown, NY 10591, USA.
Proc Natl Acad Sci U S A. 2013 Aug 20;110(34):E3179-88. doi: 10.1073/pnas.1217812110. Epub 2013 Aug 5.
Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene's transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon-intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN's artificial intron opens up engineering modalities for the generation of multifunctional alleles.
条件性突变成为研究基因功能的首选方法，但构建条件性等位基因通常很繁琐，受到靶基因结构的限制，有时还容易出现不完全条件性缺失。为了解决这些问题，我们开发了一种称为反转条件（COIN）的技术。在激活之前，COIN 包含一个倒置模块（COIN 模块），该模块惰性地位于常驻基因的反义链中。当通过位点特异性重组酶反转到 sense 链时，COIN 模块会导致靶基因转录的终止，并同时提供一个用于跟踪此事件的报告基因。COIN 模块可以插入天然内含子（内含子 COINs）或直接插入编码外显子作为人工内含子的一部分（外显子 COINs），这极大地简化了等位基因设计，并增加了对以前的条件性 KO 方法的灵活性。对 20 多个 COIN 等位基因的详细分析确立了该方法的可靠性及其广泛适用于任何基因的能力，无论其exon-intron 结构如何。我们的广泛测试提供了有助于确保该方法成功的规则，也解释了为什么其他现有的条件性方法经常不能最佳地发挥作用。最后，使用 COIN 的人工内含子分割外显子的能力为多功能等位基因的生成开辟了工程模式。
