Methods in molecular biology
-
Exon-skipping therapy is an emerging approach that uses synthetic DNA-like molecules called antisense oligonucleotides (ASOs) to splice out frame-disrupting parts of mRNA, restore the reading frame, and produce truncated yet functional proteins. Phosphorodiamidate morpholino oligomer (PMO) is one of the safest among therapeutic ASOs for patients and has recently been approved under the accelerated approval program by the US Food and Drug Administration (FDA) as the first ASO-based drug for Duchenne muscular dystrophy (DMD). Multi-exon skipping utilizing ASOs can theoretically treat 80-90% of patients with DMD. Here, we describe the systemic delivery of a cocktail of ASOs to skip exon 51 and exons 45-55 in the mdx52 mouse, an exon 52 deletion model of DMD produced by gene targeting, and the evaluation of their efficacies in vivo.
-
Post-bisulfite adaptor tagging (PBAT) is a highly efficient procedure to construct libraries for whole-genome bisulfite sequencing (WGBS). PBAT attaches adaptors to bisulfite-converted genomic DNA to circumvent bisulfite-induced degradation of library DNA inherent to conventional WGBS protocols. Consequently, it enables PCR-free WGBS from nanogram quantities of mammalian DNA, thereby serving as an invaluable tool for methylomics.
-
Exon skipping is an emerging approach to treating Duchenne muscular dystrophy (DMD), one of the most common lethal genetic disorders. Exon skipping uses synthetic antisense oligonucleotides (AONs) to splice out frame-disrupting exon(s) of DMD mRNA to restore the reading frame of the gene products and produce truncated yet functional proteins. ⋯ Although the success of multiple exon skipping in a DMD dog model has made a significant impact on the development of therapeutics for DMD, unmodified AONs such as phosphorodiamidate morpholino oligomers (PMOs) have little efficacy in cardiac muscles. Here, we describe our technique of intravenous injection of a cocktail of peptide-conjugated PMOs (PPMOs) to skip multiple exons, exons 6 and 8, in both skeletal and cardiac muscles in dystrophic dogs and the evaluation of the efficacy and toxicity.
-
Preeclampsia is a relatively common pregnancy-related condition associated with serious maternal and fetal morbidity and mortality. It is now well established that anti-angiogenic sFlt1 is upregulated in preeclampsia and binds PlGF and VEGF, causing an imbalance in angiogenic factors with subsequent endothelial injury and dysfunction. ⋯ There are several automated, commercially available immunoassays capable of measuring PlGF and the sFlt1/PlGF ratio for preeclampsia diagnosis. Here we outline the methodology for using the Roche Cobas ® e 411 immunoassay platform to determine the sFlt1/PlGF ratio.
-
Superoxide and hydrogen peroxide produced by mitochondria play important roles in various physiological and pathological processes. This chapter describes a plate-based method to measure rates of superoxide and/or hydrogen peroxide production at specific sites in isolated mitochondria.