使用pET01载体进行小基因剪接分析的实验方案。
Protocol for a minigene splice assay using the pET01 vector.
作者信息
Andreae Hannah, Curcio Marialessandra, Owrang Daniel, Esmaeelpour Sahar, Jahnke Friederike, Benseler Fritz, Brose Nils, Vona Barbara
机构信息
Institute of Human Genetics, University Medical Center Göttingen, 37073 Göttingen, Germany; Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, 37073 Göttingen, Germany; Auditory Neuroscience and Optogenetics Laboratory, German Primate Center, 37077 Göttingen, Germany.
Institute of Human Genetics, University Medical Center Göttingen, 37073 Göttingen, Germany; Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, 37073 Göttingen, Germany; Auditory Neuroscience and Optogenetics Laboratory, German Primate Center, 37077 Göttingen, Germany; Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, 80131 Naples, Italy.
出版信息
STAR Protoc. 2025 Jun 18;6(3):103908. doi: 10.1016/j.xpro.2025.103908.
Aberrant splicing plays a major role in hereditary disorders, yet characterizing molecular effects of splice variants poses challenges. Here, we present a protocol for an in vitro minigene splice assay using the pET01 vector. We describe steps for assay design, minigene plasmid cloning, transfection, RNA isolation, and cDNA synthesis. We also detail procedures for quantitative capillary electrophoresis and optional subcloning. This protocol is useful when patient RNA is unavailable or target genes or isoforms are not expressed in accessible tissues.
异常剪接在遗传性疾病中起主要作用，但表征剪接变体的分子效应面临挑战。在此，我们介绍一种使用pET01载体进行体外小基因剪接测定的方案。我们描述了测定设计、小基因质粒克隆、转染、RNA分离和cDNA合成的步骤。我们还详细说明了定量毛细管电泳和可选的亚克隆程序。当无法获得患者RNA或目标基因或异构体在可获取的组织中不表达时，该方案很有用。
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