Bezodis William, Prescott Helen, Vlad Daniela, Dickinson Hugh
Department of Biology, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK.
Bio Protoc. 2023 Aug 5;13(15):e4784. doi: 10.21769/BioProtoc.4784.
Study of gene function in eukaryotes frequently requires data on the impact of the gene when it is expressed as a transgene, such as in ectopic or overexpression studies. Currently, the use of transgenic constructs designed to achieve these aims is often hampered by the difficulty in distinguishing between the expression levels of the endogenous gene and its transgene equivalent, which may involve either laborious microdissection to isolate specific cell types or harvesting tissue at narrow timepoints. To address this challenge, we have exploited a feature of the Golden Gate cloning method to develop a simple, restriction digest-based protocol to differentiate between expression levels of transgenic and endogenous gene copies. This method is straightforward to implement when the endogenous gene contains a restriction site but, importantly, can be adapted for most genes and most other cloning strategies. Key features This protocol was developed to determine the expression level of an ectopically expressed transcription factor with broad native expression in all surrounding tissues. The method described is most directly compatible with Golden Gate cloning but is, in principle, compatible with any cloning method. The protocol has been developed and validated in the model plant but is applicable to most eukaryotes. Graphical overview.
对真核生物中基因功能的研究常常需要有关基因作为转基因表达时的影响的数据，比如在异位表达或过表达研究中。目前，用于实现这些目标的转基因构建体的使用常常受到阻碍，因为难以区分内源基因及其转基因等效物的表达水平，这可能涉及费力的显微切割以分离特定细胞类型，或者在狭窄的时间点采集组织。为应对这一挑战，我们利用了金门克隆方法的一个特点，开发了一种基于简单酶切消化的方案，以区分转基因和内源基因拷贝的表达水平。当内源基因含有一个限制性酶切位点时，该方法易于实施，但重要的是，它可以适用于大多数基因和大多数其他克隆策略。关键特性 本方案旨在确定在所有周围组织中广泛天然表达的异位表达转录因子的表达水平。所描述的方法与金门克隆最直接兼容，但原则上与任何克隆方法兼容。该方案已在模式植物中开发并验证，但适用于大多数真核生物。图形概述。
