Methods in molecular biology
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CRISPR-Cas9 has been explored as a transformative genome engineering tool for many eukaryotic organisms. However, its utilization in bacteria remains limited and ineffective. ⋯ The general principle is to use CRISPR-Cas9 as an efficient selection tool for the edited mutant (whose CRISPR-Cas9 target site has been disrupted through a homologous recombination event and thus can survive selection) against? the wild type background cells. This protocol is broadly applicable to other microorganisms for genome-editing purposes.
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DNA methylation plays an important role in the regulation of the expression of transposons and genes. Various methods have been developed to assay DNA methylation levels. Bisulfite sequencing is considered to be the "gold standard" for single-base resolution measurement of DNA methylation levels. ⋯ Here, we described a protocol for WGBS in plant species with large genomes. This protocol has been successfully applied to assay genome-wide DNA methylation levels in maize and barley. This protocol has also been successfully coupled with sequence capture technology to assay DNA methylation levels in a targeted set of genomic regions.
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Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle degeneration, caused by nonsense or frameshift mutations in the dystrophin (DMD) gene. Antisense oligonucleotides can be used to induce specific exon skipping; recently, a phosphorodiamidate morpholino oligomer (PMO) has been approved for clinical use in DMD. However, an efficient PMO delivery strategy is required to improve the therapeutic efficacy in DMD patients. ⋯ Here, we describe an efficient PMO delivery strategy using the combination of BLs and ultrasound exposure to treat muscles in a DMD mouse model (mdx). This ultrasound-mediated BL technique can increase the PMO-mediated exon-skipping efficiency, leading to significantly increased dystrophin expression. Thus, the combination of BLs and ultrasound exposure may be a feasible PMO delivery method to improve therapeutic efficacy and reduce the PMO dosage for DMD treatment.
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Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by mutations in the DMD gene. Antisense-mediated exon skipping is a promising strategy to treat DMD. The approval of Exondys 51 (eteplirsen) targeting exon 51 was the most noteworthy accomplishment in 2016. ⋯ In addition, an invasive muscle biopsy is required to obtain muscle cells from patients. Furthermore, many DMD mutations are very rare and it is hard to find a patient with a specific mutation for muscle biopsy in many cases. Here, we describe a novel approach to create an immortalized muscle cell line with a DMD deletion mutation using the human rhabdomyosarcoma (RD) cell line and the CRISPR/Cas9 system that can be used to test the efficacy of exon skipping.
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After conducting systematic and quantitative comparisons of different sample preparation techniques regarding their capability to efficiently and reproducibly recover proteins from biopsies, we present here our superior protocol for extracting proteins from low amounts of adipose tissue. Adipose tissue as a matrix in bottom-up proteomics is challenging due to the extremely high lipid content. The lysis buffer utilized contains the detergent sodium deoxycholate, which does not impair the activity of trypsin and therefore enables direct digestion without detergent removal steps. The resulting workflow is time saving, cost efficient, easy to perform, and it can also be applied to other hydrophobic samples.