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首页> 美国卫生研究院文献>Nucleic Acids Research >Yeast two-hybrid interaction partner screening through in vivo Cre-mediated Binary Interaction Tag generation
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Yeast two-hybrid interaction partner screening through in vivo Cre-mediated Binary Interaction Tag generation

机译：通过体内Cre介导的二元相互作用标签生成筛选酵母两杂交相互作用伙伴

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摘要

Yeast two-hybrid (Y2H) has been successfully used for genome-wide screens to identify protein–protein interactions for several model organisms. Nonetheless, the logistics of pair-wise screening has resulted in a cumbersome and incomplete application of this method to complex genomes. Here, we develop a modification of Y2H that eliminates the requirement for pair-wise screening. This is accomplished by incorporating lox sequences into Y2H vectors such that cDNAs encoding interacting partners become physically linked in the presence of Cre recombinase in vivo. Once linked, DNA from complex pools of clones can be processed without losing the identity of the interacting partners. Short linked sequence tags from each pair of interacting partner (binary interaction Tags or BI-Tags) are then recovered and sequenced. To validate the approach, comparisons between interactions found using traditional Y2H and the BI-Tag method were made, which demonstrate that the BI-Tag technology accurately represents the complexity of the interaction partners found in the screens. The technology described here sufficiently improves the throughput of the Y2H approach to make feasible the generation of near comprehensive interaction maps for complex organisms.
机译：酵母双杂交（Y2H）已成功用于全基因组筛选，以鉴定几种模式生物的蛋白质-蛋白质相互作用。然而，成对筛选的后勤工作导致该方法繁琐且不完整地应用于复杂基因组。在这里，我们开发了Y2H的修饰，消除了成对筛选的要求。这是通过将lox序列整合到Y2H载体中来实现的，这样，在体内存在Cre重组酶的情况下，编码相互作用伴侣的cDNA就会物理连接。一旦链接，就可以处理来自复杂克隆池的DNA，而不会失去相互作用伙伴的身份。然后从每对相互作用的伴侣对中获得短链序列标签（二进制相互作用标签或BI标签）并进行测序。为了验证该方法，对使用传统Y2H和BI-Tag方法发现的交互进行了比较，这表明BI-Tag技术准确地代表了屏幕中发现的交互伙伴的复杂性。这里描述的技术充分提高了Y2H方法的通量，从而使复杂生物的近乎全面的相互作用图的生成成为可能。

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