Project description:In this study, in order to minimize the genetic variability of muscle samples we have used two approaches. First, we have analyzed the gene expression profile from a single skeletal muscle, the medial gastrocnemius (MG), and not from a pool of different muscles which could have different expression profiles. Second, we have performed the temporal gene expression profiling by extracting the MG muscles of the same individual from the both legs at two different times to minimize the inter-individual genetic variability. The MG muscle represent an excellent candidate for biopsy due to its easy accessible by surgery and also because its biopsy is well tolerated by the animals allowing us to perform another later biopsy in the other leg to obtain two MG samples of the same individual at two different times. Moreover, MG muscle is composed of approximately 20-30% type I (red) fibers and 70-80% type II (white) fibers {Ariano MA, 1973}, {Simard C, 1988}, {Zhan WZ, 1992} and therefore is more representative of skeletal muscle tissue in general than a muscle composed exclusively by red or white fibers. The transcript expression profiles in MG muscles from mdx and wild-type mice were analyzed at 3 weeks, 1.5 months and 3 months of life by using the 430 2.0 gene chips from Affymetrix (n=3 for each condition). The differentially expressed transcripts which showed differences ?1.5-fold were obtained by performing three different comparisons: 1) genes differentially expressed in mdx compared with controls at each point in time (additional file 1); 2) temporal analysis of the genes differentially expressed in mdx mice between the three points in time also compared with the variations in control mice (additional file 2); and 3) temporal analysis of the genes differentially expressed in control mice between the three points in time also compared with the variations in mdx mice (additional file 3). The first comparison that we performed, by comparing the gene expression between mdx and control mice at every point in time, was similar to that performed in previous longitudinal studies {Porter JD, 2003}, {Rouger K, 2002}, {Turk R, 2005}. However, the other two comparisons were directed to elucidate the genes that are varying throughout the period of time analyzed in every mice strain, and therefore we obtained on the one hand the genes that vary in mdx mice but not in wild-type, and on the other hand the genes that vary in control animals but remain unchanged in mdx mice between the times analyzed. To present the results in a more comprehensive form, all the genes were classified in seven different categories: Cell adhesion & extracellular matrix; Proteolysis; Muscle structure & regeneration; Inflammation & immune response; Cell signaling & cell communication; Metabolism; and Others/unknown. The resulting genes from our study were classified in their functional categories using information from Affymetrix (www.affymetrix.com) and from the Gene Ontology database accessible in the Jackson Laboratory Mouse Genome Informatics website (www.informatics.jax.org).

Project description:Transgenic overexpression of Galgt2 in the skeletal muscles of mdx mice inhibits the development of disease pathology associated with muscular dystrophy. This is the case both in transgenic mice, where Galgt2 overexpression occurs from embryonic timepoints onward and in mdx mice where Galgt2 is overexpressed in the early postnatal period using Adeno-associated virus (AAV). Here, we use gene expression profiling to compare transcriptional changes resulting from embryonic and postnatal Galgt2 overexpression in mdx skeletal muscle. A surprising number of changes were in genes known to ameliorate muscular dystrophy when overexpressed (agrin, integrin alpha 7, ADAM12, Bcl2) or to cause muscular dystrophy when mutated (collagen VI (alpha1,alpha2), plectin 1, dystroglycan, selenoprotein N1, integrin alpha7, biglycan, dysferlin). Several genes involved in calcium homeostasis were also changed. In Galgt2 transgenic mice, where embryonic overexpression of Galgt2 in skeletal muscles alters neuromuscular development and muscle growth, the number of gene expression changes was vastly greater, however, 14% of genes altered in postnatal AAV-Galgt2 infected mdx muscles were also changed with embryonic overexpression. These experiments suggest that postnatal overexpression of Galgt2 inhibits muscular dystrophy in mdx mice via induction of a group of genes that, in aggregate, can govern membrane stability, membrane repair, calcium homeostasis, and apoptosis. Transgenic overexpression of Galgt2 in the skeletal muscles of mdx mice inhibits the development of disease pathology associated with muscular dystrophy. This is the case both in transgenic mice, where Galgt2 overexpression occurs from embryonic timepoints onward and in mdx mice where Galgt2 is overexpressed in the early postnatal period using Adeno-associated virus (AAV). Here, we use gene expression profiling to compare transcriptional changes resulting from embryonic and postnatal Galgt2 overexpression in mdx skeletal muscle. A surprising number of changes were in genes known to ameliorate muscular dystrophy when overexpressed (agrin, integrin alpha7, ADAM12, Bcl2) or to cause muscular dystrophy when mutated (collagen VI (alpha1,alpha2), plectin 1, dystroglycan, selenoprotein N1, integrin alpha7, biglycan, dysferlin). Several genes involved in calcium homeostasis were also changed. In Galgt2 transgenic mice, where embryonic overexpression of Galgt2 in skeletal muscles alters neuromuscular development and muscle growth, the number of gene expression changes was vastly greater, however, 14% of genes altered in postnatal AAV-Galgt2 infected mdx muscles were also changed with embryonic overexpression. These experiments suggest that postnatal overexpression of Galgt2 inhibits muscular dystrophy in mdx mice via induction of a group of genes that, in aggregate, can govern membrane stability, membrane repair, calcium homeostasis, and apoptosis. Experiment Overall Design: Microarrays were done in three groups at a time. 1) Transgenic Overexpression of Galgt2 - comprised to Wild Type (WT), Galgt2 Transgenic (CT), Dystrophin-Deficient (mdx), and Galgt2 Transgenic and Dystrophin-Deficient (CTmdx) each in duplicate 2) AAV-Mediated Galgt2 Gene Delivery in to the mdx gastrocnemius muscle in the postnatal period - comprised of AAVGalgt2 and PBS each in duplicate, and 3) Myoblasts and myotubes - comptised of one sample each of C1C12 myoblasts and myotubes, C2C12 myoblasts and myotubes stably transfected with Galgt2

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Transcription profiling of mouse leg muscle mdx vs. control - time series

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