Methods in molecular biology
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DNA methylation profiling in the epigenome of Arabidopsis thaliana (Arabidopsis) has provided great insights in the role of this epigenetic mark for the regulation of transcription in plants, and is often based on high-throughput sequencing. The analysis of these data involves a series of steps including quality checks, filtering, alignment, identification of methyl-cytosines, and the identification of differentially methylated regions. This chapter outlines the computational methodology required to profile genome-wide differential methylation patterns based on publicly available Arabidopsis base-resolution bisulfite sequencing data. The methylPipe Bioconductor package is adopted for the identification of the differentially methylated regions, and all the steps from the raw data to the required input are described in detail.
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Exon skipping therapy using synthetic DNA-like molecules called antisense oligonucleotides (ASOs) is a promising therapeutic candidate for overcoming the dystrophin mutation that causes Duchenne muscular dystrophy (DMD). This treatment involves splicing out the frame-disrupting segment of the dystrophin mRNA, which restores the reading frame and produces a truncated yet functional dystrophin protein. Phosphorodiamidate morpholino oligomer (PMO) is the safest ASO for patients among ASOs and has recently been approved under the accelerated approval pathway by the U. ⋯ Food and Drug Administration (FDA) as the first drug for DMD. Here, we describe the methodology and protocol of PMO transfection and evaluation of the exon skipping efficacy in the mdx52 mouse, an exon 52 deletion model of DMD produced by gene targeting. The mdx52 mouse model offers advantages over the mdx mouse, a spontaneous DMD model with a nonsense mutation in exon 23, in terms of the deletion in a hotspot of deletion mutations in DMD patients, the analysis of caveolae and also Dp140 and Dp260, shorter dystrophin isoforms.
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Spinal muscular atrophy (SMA) is the most common genetic cause of infantile death caused by mutations in the SMN1 gene. Nusinersen (Spinraza), an antisense therapy-based drug with the 2'-methoxyethoxy (2'MOE) chemistry approved by the FDA in 2016, brought antisense drugs into the spotlight. ⋯ To investigate new chemistries of antisense oligonucleotides (ASOs), SMA mouse models can serve as an important source. Here we describe methods to test the efficacy of ASOs, such as phosphorodiamidate morpholino oligomers (PMOs), in a severe SMA mouse model.
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Antisense oligonucleotides (AONs) have been actively developed for more than 30 years as a form of molecular medicine and represent promising therapeutic tools for many disorders. Significant progress has been made toward their clinical development in particular for splice switching AONs for the treatment of neuromuscular disorders such as Duchenne muscular dystrophy (DMD). Many different chemistries of AONs can be used for splice switching modulation, and some of them have now reached regulatory approval. ⋯ Here we describe the methods to evaluate the potency of tricyclo-DNA (tcDNA)-AONs, a novel class of AONs which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration (Goyenvalle et al., Nat Med 21:270-275, 2015; Goyenvalle et al., J Neuromuscul Dis 3:157-167, 2016; Relizani et al., Mol Ther Nucleic Acids 8:144-157, 2017; Robin et al., Mol Ther Nucleic Acids 7:81-89, 2017). We will focus on the preclinical evaluation of these tcDNA for DMD, specifically targeting the exon 51 of the human dystrophin gene. We will first detail methods to analyze their efficacy both in vitro in human myoblasts and in vivo in the hDMD and mdx52 mouse models and then describe means to evaluate their potential renal toxicity.
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Reactive oxygen species (ROS) are a group of unstable and highly reactive molecules or free radicals typically generated as by-products of cellular processes involving molecular oxygen. In vascular cells, the excessive ROS generation results in the initiation and progression of cardiovascular diseases (CVD). Therefore, a dynamic, robust, and accurate ROS detection method in the blood vessels is essential for pathophysiological research studies of the cardiovascular system. ⋯ The protocol includes preparation of frozen aortic tissue sections, monitoring DHE oxidation-derived fluorescence by fluorescence microscopy, and high-performance liquid chromatograph-based analysis of MitoSOX and its oxidation products. For studying the role of AMP-activated protein kinase (AMPK) in the redox regulation, we employed AMPKα2 knockout mice and observed increased superoxide and mitochondrial superoxide levels in the aorta of AMPK knockout mice relative to the wild-type group. This novel ROS detection method will be valuable for investigating the roles of cellular and/or mitochondrial ROS in the pathogenesis of CVDs.