This proposal seeks to establish an assay for factors provoking limb regeneration in a model organism, which will inform parallel efforts to design methods for enabling muscle, skeletal structures or even whole limbs of human beings to regenerate. Tadpoles of Xenopus laevis can regenerate limbs at early developmental stages but not at later stages. The rationale for the current proposal is whatever regenerative mechanism is lost is probably missing in mammalian limbs and responsible for their inability to regenerate: limb fibroblasts can be re-specified to a limb bud phenotype by specific transcription factors. A selective system will isolate cells that have been appropriately re-specified, and performance of the regenerated muscle fibers, discrete muscles, and entire regenerated appendages will be assessed quantitatively with the help of the Muscle Force Assessment Core. This project uses methods that have seldom, if ever, been used to study muscle development, differentiation and regeneration: iPS technology to re-define cells;tissue culture methods to investigate regeneration;use of flow cytometry to select the desired cells.
Specific Aims of the proposal are:
Specific Aim 1; To respecify froglet limb-derived fibroblasts to a. limb bud state. The working hypothesis is that this can be achieved by introduction of genes encoding the limb bud state, together with genes to unwrap the chromatin, followed by a selective step for positively responding cells.
Specific Aim 2 : To rescue regenerative ability in Xenopus froglet limbs by re-introduction of limb bud cells. The working hypothesis is that this can be achieved by injecting limb bud cells subcutaneously, allowing to heal, and amputation through the graft region.
This Core Center will develop disease specific and mutation specific progenitor cells that will be of value in determining effects on cell differentiation and development in identified muscle diseases.
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