Muscle Gene Therapy: Methods and Protocols by Charis L. Himeda, Xiaolan Chen, Stephen D. Hauschka (auth.),

By Charis L. Himeda, Xiaolan Chen, Stephen D. Hauschka (auth.), Dongsheng Duan (eds.)

Gene treatment bargains many conceptual merits to regard muscle ailments, particularly a number of kinds of muscular dystrophies; notwithstanding, it faces a few precise demanding situations, together with the necessity to bring a healing vector to all muscle groups in the course of the physique. In Muscle Gene treatment: tools and Protocols, professional researchers within the box current a suite of strategies geared toward bridging the translational hole in muscle gene remedy among the commonplace rodent versions and very important better animal types. Divided into 3 sections, this quantity examines simple protocols for optimizing the muscle gene expression cassette and for comparing the healing results, new advancements in muscle gene remedy know-how equivalent to adeno-associated viral vector (AAV), oligonucleotide-mediated exon-skipping, and novel RNA-based techniques, and step by step advice on muscle gene supply in swine, ovine, canines, and non-human primates. Written within the hugely profitable tools in Molecular Biology™ sequence layout, chapters comprise introductions to their respective themes, lists of the mandatory fabrics and reagents, distinctive, conveniently reproducible laboratory protocols, and pointers on troubleshooting and warding off recognized pitfalls. Authoritative and state of the art, Muscle Gene remedy: tools and Protocols serves as a useful source for graduate scholars, post-doctoral fellows, and precept investigators pursuing the an important development of muscle sickness gene treatment within the wish of sometime curing those debilitating disorders.

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Benzonase (Sigma). DNAse I: 10 mg/mL in H2O. 0), 1% (w/v) SDS. Store at room temperature. Proteinase K (Sigma). Glycogen (Invitrogen, UK). 3 M Na3 citrate. Hybridization buffer (Amersham, UK). 5× SSC). Store at 4°C. 1×).  1. De novo synthesis of functional dystrophin molecules. Dystrophin is a rod-like protein of 3,685 amino acids (aa) localized beneath the inner surface of muscle cell membrane. It functions through four major structural domains: a N-terminal domain (1-756 aa), a central rod domain (757-3122 aa), a cysteine-rich (CR) domain (3123-3409 aa), and a distal C-terminal domain (3410-3685 aa).

Methods Mol Biol 290, 281–304. 22. Sternberg, E. , Perry, W. , and Olson, E. N. (1988) Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene. Mol Cell Biol 8, 2896–2909. 23. Johnson, J. , Wold, B. , and Hauschka, S. D. (1989) Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice. Mol Cell Biol 9, 3393–3399. 24. Jaynes, J. , Johnson, J.

Full sequence data and corresponding files are normally provided in CD-ROM or e-mail for the customer following the final assembly of the de novo synthesized oligonucleotide sequences after verification of the final construct by sequencing. De novo synthesized species/codonoptimized cDNAs were supplied by GENEART as lyophilized plasmid material, reconstituted in water upon arrival and further downstream processed as required. Gene optimization and gene synthesis services are normally based on a pure fee-for-service model.

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