Methods in molecular biology
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Breakdown of the blood-brain barrier (BBB) is present in several neurological disorders such as stroke, brain tumors, and multiple sclerosis. Noninvasive evaluation of BBB breakdown is important for monitoring disease progression and evaluating therapeutic efficacy in such disorders. One of the few techniques available for noninvasively and repeatedly localizing and quantifying BBB damage is magnetic resonance imaging (MRI). ⋯ The accuracy and reliability of two of these multiparametric MRI measures, CBF by AST and DCE-MRI determined influx of Gd-DTPA, have been established by nearly congruent quantitative autoradiographic (QAR) studies with appropriate radiotracers. In addition, some of their linkages to local pathology have been shown via corresponding light microscopy and fluorescence imaging. This chapter describes: (1) multiparametric MRI techniques with emphasis on DCE-MRI and AST-MRI; (2) the measurement of the blood-to-brain influx rate constant and CBF; and (3) the role of each in determining BBB permeability.
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Stem cell culture systems that rely on undefined animal-derived components introduce variability to the cultures and complicate their therapeutic use. The derivation of human embryonic stem cells and the development of methods to produce induced pluripotent stem cells combined with their potential to treat human diseases have accelerated the drive to develop xenogenic-free, chemically defined culture systems that support pluripotent self-renewal and directed differentiation. In this chapter, we describe four xeno-free culture systems that have been successful in supporting undifferentiated growth of hPSCs as well as methods for xeno-free subculture and cryopreservation of hPSCs. Each culture system consists of a xeno-free growth medium and xeno-free substratum: (1) TeSR2™ with human recombinant laminin (LN-511); (2) NutriStem™ with LN-511; (3) RegES™ with human foreskin fibroblasts (hFFs); (4) KO-SR Xeno-Free™/GF cocktail with CELLstart™ matrix.
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Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted, Becker muscular dystrophy (BMD)-like, but functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript so that gene expression is restored. ⋯ However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, and in particular phosphorodiamidate morpholino oligomers (PMOs), for the targeted skipping of specific exons on the DMD gene.
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Exon skipping is currently one of the most promising molecular therapies for Duchenne muscular -dystrophy (DMD). We have recently developed multiple exon skipping targeting exons 6 and 8 in -dystrophin mRNA of canine X-linked muscular dystrophy (CXMD), an animal model of DMD, which exhibits severe dystrophic phenotype in skeletal muscles and cardiac muscle. We have induced efficient exon skipping both in vitro and in vivo by using cocktail antisense 2'O-methyl oligonucleotides (2'OMePS) and cocktail phosphorodiamidate morpholino oligomers (morpholinos, or PMOs) and ameliorated phenotype of dystrophic dogs by systemic injections. The multiple exon skipping (double exon skipping) shown here provides the prospect of choosing deletions that optimize the functionality of the truncated dystrophin protein for DMD patients by using a common cocktail that could be validated as a single drug and also potentially applicable for more than 90% of DMD patients.
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The primary function of skeletal muscle is to generate force. Muscle force production is compromised in various forms of acquired and/or inherited muscle diseases. An important goal of muscle gene therapy is to recover muscle strength. ⋯ These include ex vivo and in situ analysis of the contractile profile of a single intact limb muscle (the extensor digitorium longus for ex vivo assay and the tibialis anterior muscle for in situ assay), grip force analysis, and downhill treadmill exercise. Force measurement in a single muscle is extremely useful for pilot testing of new gene therapy protocols by local gene transfer. Grip force and treadmill assessments offer body-wide evaluation following systemic muscle gene therapy.