Methods in molecular biology
-
DNA sensing by the STING pathway is emerging to be a crucial component of the antitumor immune response. Although it plays a key role in the activation of tumor immune cells, exactly how STING is activated by tumor cells is not fully understood. ⋯ Here we describe a simple coculture protocol allowing for the functional characterization of cGAS/STING activity in tumor cells, together with cGAMP transfer to adjacent cells. This approach will help define how different tumors engage the STING pathway, and whether synthetic STING agonists should be used to potentiate the antitumor effects of chemotherapies.
-
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.
-
The discovery and adaptation of RNA-guided nucleases has resulted in the rapid development of efficient, scalable, and easily accessible synthetic biology tools for targeted genome editing and transcriptional control. In these systems, for example CRISPR-Cas9 from Streptococcus pyogenes, a protein with nuclease activity is targeted to a specific nucleotide sequence by a short RNA molecule, whereupon binding it cleaves the targeted nucleotide strand. To extend this genome-editing ability to the industrially important oleaginous yeast Yarrowia lipolytica, we developed a set of easily usable and effective CRISPR-Cas9 episomal vectors. ⋯ A second method demonstrates how the same CRISPR-Cas9 system can be used to induce markerless gene cassette integration into the genome by inducing homologous recombination after DNA cleavage by Cas9. Finally, we describe how a catalytically inactive form of Cas9 fused to a transcriptional repressor can be used to control transcription of native genes in Y. lipolytica. The CRISPR-Cas9 tools and strategies described here greatly increase the types of genome editing and transcriptional control that can be achieved in Y. lipolytica, and promise to facilitate more advanced engineering of this important oleaginous host.
-
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.
-
Comparative Study
Comparison of Mitochondrial Incubation Media for Measurement of Respiration and Hydrogen Peroxide Production.
High-Resolution FluoRespirometry is a well-established and versatile approach to study mitochondrial oxygen uptake amperometrically in combination with measurement of fluorescence signals. One of the most frequently applied fluorescent dyes is Amplex UltraRed for monitoring rates of hydrogen peroxide production. Selection of an appropriate mitochondrial respiration medium is of crucial importance, the primary role of which is to support and preserve optimum mitochondrial function. ⋯ Stability of assay sensitivity over experimental time was highest in MiR05 but slightly less in MiR07. Taken together, MiR05 and Buffer Z yield comparable results on respiration and H2O2 production. Despite the lower sensitivity, MiR05 was selected as the medium of choice for FluoRespirometry due to the highest stability of the sensitivity or calibration constant observed in experiments over periods of up to 2 h.