Modified whole-mount in situ hybridization protocol for the detection of transgene expression in electroporated chick embryos.
Modified whole-mount in situ hybridization protocol for the detection of transgene expression in electroporated chick embryos.
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DOI：
10.1371/journal.pone.0002638
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发表时间：
2008-07-09
期刊：
影响因子：
3.7
通讯作者：
Tavares AT
中科院分区：
文献类型：
--
作者：
Arede N;Tavares AT
体内电穿孔已被广泛用作 DNA 转移的有效手段，用于分析基因功能以及发育系统中的基因调控。在这两种类型的研究中，电穿孔转基因的正确空间和时间表达只能通过原位杂交来准确评估。在分析电穿孔鸡胚胎中的转基因表达时，我们验证了当通过传统的整体原位杂交（WISH）处理胚胎时，转基因核糖核酸探针与外源质粒DNA交叉杂交。在这里，我们描述了对 WISH 协议的修改，这对于防止 DNA 交叉杂交和特异性检测电穿孔胚胎中的转基因 mRNA 转录本至关重要。我们优化的 WISH 程序不仅适用于电穿孔鸡胚，还适用于已转染大量报告基因或表达构建体 DNA 的其他胚胎或成体组织。
In vivo electroporation has been extensively used as an effective means of DNA transfer for analyzing gene function as well as gene regulation in developmental systems. In any of these two types of studies, the correct spatial and temporal expression of the electroporated transgene can only be accurately assessed by in situ hybridization. While analyzing transgene expression in electroporated chicken embryos, we verified that transgene riboprobes cross-hybridized with the exogenous plasmid DNA when embryos were processed by conventional whole-mount in situ hybridization (WISH). Here we describe a modification to the WISH protocol that is essential to prevent DNA cross-hybridization and to specifically detect transgene mRNA transcripts in electroporated embryos. Our optimized WISH procedure can be applied not only to electroporated chick embryos but also to other embryos or adult tissues that have been transfected with large amounts of reporter- or expression construct DNA.
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影响因子：
21.3
作者：
Itasaki, N;Bel-Vialar, S;Krumlauf, R
通讯作者：
Krumlauf, R
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Saito, T;Nakatsuji, N
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Nakatsuji, N
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Tavares, Ana Teresa;Andrade, Sofia;Belo, Jose Antonio
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Belo, Jose Antonio
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HENRIQUE, D;ADAM, J;ISHHOROWICZ, D
通讯作者：
ISHHOROWICZ, D