Methods in molecular biology
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Hair follicle stem cells (HFSCs) are noted for their relative quiescence and therefore can be distinguished from other cells by their differential history of cell division. Replicating cells can be labeled by pulsing the animals repeatedly with 5-bromo-2'-deoxyuridine (BrdU) or tritiated thymidine ([3H]TdR), thymidine analogs that get incorporated into DNA during DNA synthesis. ⋯ Alternatively, a well-established tet-regulatable transgenic mouse model can be used to express histone H2B-GFP in epithelial proliferative cells and their dilution and retention of the GFP signal can be followed. In this chapter, we detail the steps to perform BrdU pulse-chase and H2B-GFP pulse-chase experiments to identify quiescent cells in the hair follicle.
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The epigenetic mark 5-methylcytosine confers heritable regulation of gene expression that can be dynamically modulated during transitions in cell fate. With the development of high-throughput sequencing technologies, it is now possible to obtain comprehensive genome-wide maps of the mammalian DNA methylation landscape, but the application of these techniques to limited material remains challenging. ⋯ In this strategy, bisulfite treatment is performed prior to library generation in order to both convert unmethylated cytosines and fragment DNA to an appropriate size. Then sequencing adapters are added by complementary strand synthesis using random tetramer priming, and libraries are subsequently amplified by PCR.
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The application of antisense oligonucleotides (AONs) to modify pre-messenger RNA splicing has great potential for treating genetic diseases. The strategies used to redirect splicing for therapeutic purpose involve the use of AONs complementary to splice motifs, enhancer or silencer sequences. AONs to block intronic splicing silencer motifs can efficiently augment exon 7 inclusion in survival motor neuron 2 (SMN2) gene and have demonstrated robust therapeutic effects in both preclinical studies and clinical trials in spinal muscular atrophy (SMA), which has led to a recently approved drug. ⋯ Here we provide the details of methods that our lab has used to evaluate PMO-mediated SMN2 exon 7 inclusion in the in vivo studies conducted in SMA transgenic mice. The methods comprise mouse experiment procedures, assessment of PMOs on exon 7 inclusion at RNA levels by reverse transcription (RT-) PCR and quantitative real-time PCR. In addition, we present methodology for protein quantification using western blot in mouse tissues, on neuropathology assessment of skeletal muscle (muscle pathology and neuromuscular junction staining) as well as behaviour test in the SMA mice (righting reflex).
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Animal spinal cord injury (SCI) models have proven highly useful for investigating the mechanisms involved in the injury process and evaluating the effectiveness of experimental therapeutic interventions. Over the last years, substantial improvements have been made in producing consistent and reproducible animal SCI models. Different SCI models have been developed to address the mechanism of injury, being divided into contusion, compression, distraction, dislocation, transection, or chemical models. The method described here is a mouse compression model of SCI that, in many respects, faithfully reproduces SCI in man.
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Over the past three decades, attempts at understanding the multifaceted mechanisms underlying the pathophysiology of traumatic brain injury (TBI) have seen the development of numerous animal models to investigate changes in molecular and cellular pathways and neurobehavioral outcomes. Until now, controlled cortical impact (CCI) represents the most frequently used mechanical model to induce TBI, given its accuracy, easy of control, and, most importantly, its ability to produce brain injuries similar to those seen in humans. The CCI model is based on the use of an impact system that delivers a physical impact to the exposed dura of an animal. This chapter will describe in detail the electromagnetic CCI model of TBI in mice.