共查询到20条相似文献,搜索用时 15 毫秒
1.
R. A. Shafei I. A. Zelenina M. L. Semenova V. A. Kolesnikov V. M. Mikhailov V. S. Baranov A. V. Zelenin V. A. Golichenkov 《Russian Journal of Developmental Biology》2000,31(5):326-331
We used the method of particle bombardment (ballistic transfection) to introduce β-galactosidase and human dystrophin genes
into mouse embryos and skeletal muscles of adult mice. We examined the mechanisms of DNA transfer into skeletal muscle cells,
the biological processes accompanying and following this transfer, the susceptibility of various types of muscle cells to
transfection, and the duration of expression of and conditions affecting the introduced genes. We have also developed an effective,
convenient, and practical methods of skeletal muscle transfection. 相似文献
2.
In vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic delivery of therapeutic proteins. Electrotransfer is a powerful method for DNA transfer into skeletal muscle. In view of the broad potential gene therapy clinical application of electrotransfer offers, it is important to perform toxicology studies on electrotransfered muscle tissue. We have investigated if the delivery of square wave electric pulses of low field strength and long duration to mouse tibial cranial muscle induced the expression of stress related genes. We have profiled gene expression patterns in muscles at different times after delivery of electric pulses using Stress/Toxicology microarrays. No significant variation in the expression of stress related-genes was detected between treated and non-treated muscles. This suggests that application of adequate, fine-tuned, electric pulses to the skeletal muscle is a non-toxic technique for gene therapy. 相似文献
3.
Wang Z Zhu T Qiao C Zhou L Wang B Zhang J Chen C Li J Xiao X 《Nature biotechnology》2005,23(3):321-328
Systemic gene delivery into muscle has been a major challenge for muscular dystrophy gene therapy, with capillary blood vessels posing the principle barrier and limiting vector dissemination. Previous efforts to deliver genes into multiple muscles have relied on isolated vessel perfusion or pharmacological interventions to enforce broad vector distribution. We compared the efficiency of multiple adeno-associated virus (AAV) vectors after a single injection via intraperitoneal or intravenous routes without additional intervention. We show that AAV8 is the most efficient vector for crossing the blood vessel barrier to attain systemic gene transfer in both skeletal and cardiac muscles of mice and hamsters. Serotypes such as AAV1 and AAV6, which demonstrate robust infection in skeletal muscle cells, were less effective in crossing the blood vessel barrier. Gene expression persisted in muscle and heart, but diminished in tissues undergoing rapid cell division, such as neonatal liver. This technology should prove useful for muscle-directed systemic gene therapy. 相似文献
4.
Skeletal muscle is a promising target tissue for the gene therapy of both muscle and non-muscle disorders. Gene transfer into muscle tissue can produce a variety of physiologically active proteins and may ultimately be applied to the treatment of many diseases. A variety of methods have been studied to transfer genes into skeletal muscle, including viral and non-viral vectors. In this review, we discuss recent developments in the non-viral delivery of genes to muscles. 相似文献
5.
Differential expression of two MyoD genes in fast and slow muscles of gilthead seabream ( Sparus aurata) 总被引:2,自引:0,他引:2
Members of the myogenic regulatory gene family, including MyoD, Myf5, Myogenin and MRF4, are specifically expressed in myoblast and skeletal muscle cells and play important roles in regulating skeletal muscle development and growth. They are capable of converting a variety of non-muscle cells into myoblasts and myotubes. To better understand their roles in the development of fish muscles, we have isolated the MyoD genomic genes from gilthead seabream (Sparus aurata), analyzed the genomic structures, patterns of expression and the regulation of muscle-specific expression. We have demonstrated that seabream contain two distinct non-allelic MyoDgenes, MyoD1 and MyoD2. Sequence analysis revealed that these two MyoD genes shared a similar gene structure. Expression studies demonstrated that they exhibited overlapping but distinct patterns of expression in seabream embryos and adult slow and fast muscles. MyoD1 was expressed in adaxial cells that give rise to slow muscles, and lateral somitic cells that give rise to fast muscles. Similarly, MyoD2 was initially expressed in both slow and fast muscle precursors. However, MyoD2 expression gradually disappeared in the adaxial cells of 10- to 15-somite-stage embryos, whereas its expression in fast muscle precursor cells was maintained. In adult skeletal muscles, MyoD1 was expressed in both slow and fast muscles, whereas MyoD2 was specifically expressed in fast muscles. Treating seabream embryos with forskolin, a protein kinase A activator, inhibited MyoD1 expression in adaxial cells, while expression in fast muscle precursors was not affected. Promoter analysis demonstrated that both MyoD1 and MyoD2 promoters could drive green fluorescence protein expression in muscle cells of zebrafish embryos. Together, these data suggest that the two non-allelic MyoD genes are functional in seabream and their expression is regulated differently in fast and slow muscles. Hedgehog signaling is required for induction of MyoDexpression in adaxial cells. 相似文献
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阳离子脂质体转染人类骨骼肌原代细胞的初步研究 总被引:1,自引:0,他引:1
探讨不同脂质体介导基因转染人类骨骼肌原代细胞的转染效率和基因的表达.将含有β-半乳糖苷酶LacZ结构基因的质粒,用三种不同的阳离子脂质体导入人类骨骼肌原代细胞中,通过X-Gal染色观察不同的转染效率.结果发现,Fugene 6转染效率最高,蓝染细胞达10%,其脂质体与DNA的最佳比例为3∶ 2.Fugene 6可有效地将外源基因导入骨骼肌原代细胞,而且外源基因可以长效高效地表达,有望用来作为基因治疗的载体. 相似文献
8.
Direct gene transfer and expression into rat heart in vivo 总被引:18,自引:0,他引:18
We found previously that genes injected into skeletal muscle can be taken up by myofibers and expressed. In the present study we found that myocardial cells can also express a variety of reporter genes injected into myocardium as efficiently as skeletal myofibers, while the cells of several other tissues cannot. The inability of tissues other than striated muscle to express injected DNA is not due to technical difficulties of injection because injected DNA was detected in these other tissues by PCR analysis. These results suggest that skeletal and cardiac muscle cells have unique features such as T tubules that may play a critical role in DNA uptake. Expression in cardiac muscle was stable for only two weeks, possibly because of an immune response against the transfected cells. The ability to directly transfer genes into myocardial cells raises the possibility of gene therapy for both acquired and genetic heart diseases. 相似文献
9.
Kusakabe R Takechi M Tochinai S Kuratani S 《Journal of experimental zoology. Part B. Molecular and developmental evolution》2004,302(2):121-133
Agnathan lampreys retain ancestral characteristics of vertebrates in the morphology of skeletal muscles derived from two mesodermal regions: trunk myotomes and unsegmented head mesoderm. During lamprey development, some populations of myoblasts migrate via pathways that differ from those of gnathostomes. To investigate the evolution of skeletal muscle differentiation in vertebrates, we characterize multiple contractile protein genes expressed in the muscle cells of the Japanese lamprey, Lethenteron japonicum. Lamprey actin gene LjMA2, and myosin heavy chain (MyHC) genes LjMyHC1 and LjMyHC2 are all expressed in the developing skeletal muscle cells of early embryos. However, LjMyHC1 and LjMyHC2 are expressed only in cells originating from myotomes, while LjMA2 is expressed in both myotomal and head musculature. Thus, in lampreys, myotomes and head mesoderm differ in the use of genes encoding contractile protein isoforms. Phylogenetic tree analyses including lamprey MyHCs suggest that the variety of muscle MyHC isoforms in different skeletal muscles may correspond to the morphological complexity of skeletal muscles of different vertebrate species. Another lamprey actin gene LjMA1 is likely to be the first smooth muscle actin gene isolated from non-tetrapods. We conclude that, in vertebrate evolution, the different regulatory systems for striated and smooth muscle-specific genes may have been established before the agnathan/gnathostome divergence. 相似文献
10.
Gene transfer techniques possess tremendous potential for treating diseases and for facilitating the study of basic physiological processes. However, further development of efficient and safe methods for gene transfer is needed. The purpose of this study was to test the hypothesis that mechanical strain increases the transfer of DNA to differentiated skeletal muscle cells. We tested this hypothesis by applying cyclic strain to cultured skeletal myotubes either prior to or immediately after the introduction of exogenous DNA complexed with lipids, with strains of varying magnitude (10%, 20% and 30%), number (1800, 3600 and 7200 strain cycles) and frequency (0.5, 1.0 and 1.5 Hz). Results demonstrated that DNA transfection was increased by exposing muscle cells to cyclic strain, and that strain magnitude, number and frequency each influenced DNA transfection. Optimal strain conditions (20% strain magnitude, 3600 cycles applied at 1 Hz) were utilized to examine the role of membrane transport systems in strain-induced increases in DNA transfection. Filipin III was used to inhibit caveolar transport and was found to inhibit strain-mediated increases in DNA transfection, whereas chlorpromazine, used to inhibit clathrin-coated vesicle transport, had no effect. These results indicate that mechanical strain may be an effective method for increasing DNA transfection in skeletal muscle through enhanced caveolar transport. 相似文献
11.
Peng B Zhao Y Lu H Pang W Xu Y 《Biochemical and biophysical research communications》2005,338(3):1490-1498
In vivo plasmid DNA electroporation resulted in elevated and lasting transgene expression in skeletal muscles. But the nature of the cells that contributed to sustained gene expression remains unknown. We followed the fate of plasmid DNA delivered with electroporation and systematically investigated the time course and location of transgene expression in muscle tissues both with GFP and luciferase. Furthermore, satellite cell activation after electroporation was confirmed by RT-PCR and immunohistochemistry analysis. The activated satellite cells were shown to be able to uptake the injected plasmid DNA and express transgene products as regenerated myocytes. We found that cells with longer gene expression durations were mostly regenerated muscle fibers. In contrast, expression in pre-existing muscle fibers was rather transient. We also presented in this study that immune response to transgene products might hamper the lasting gene expression. Based on these observations, we proposed that the underlying mechanism for prolonged transgene expression in the muscles after electroporation is related to the activation and transfection of myogenic satellite cells which subsequently developed into regenerated muscle fibers. 相似文献
12.
We have isolated an avian muscle cell line (QM) which has the essential features of established mammalian muscle cell lines. The experiments reported here were undertaken to determine the suitability of QM cells for the introduction and analysis of cloned transgenes. The promoter of the cardiac troponin T (cTNT) gene has been previously shown to contain sequence elements which govern muscle-specific expression of the chloramphenicol acetyltransferase (CAT) gene in transiently transfected primary cell cultures. We show here that QM cells stably harboring cTNT promoter-CAT fusion genes up-regulate CAT expression in concert with myogenic differentiation, and that as few as 110 upstream nucleotides are sufficient for such differentiation-dependent regulation. In addition, both transient and stable transfection experiments demonstrate that differentiated QM cells possess trans-acting factors necessary for the expression of the skeletal alpha-actin promoter, despite the absence of mRNA or protein product from the endogenous sarcomeric actin genes in these cells. Finally, to follow the developmental potential of QM cells in vivo, we created a clone, QM2ADH, which constitutively expresses the histochemical marker transgene encoding Drosophila alcohol dehydrogenase. When surgically inserted into the limb buds of developing chick embryos, QM2ADH cells are incorporated into endogenous developing muscles, indicating that QM cells are capable of recognizing and responding to host cues governing muscle morphogenesis. Thus, QM cells are versatile as recipients of transgenes for the in vitro and in vivo analysis of molecular events in muscle development. 相似文献
13.
Liposome-Mediated Gene Transfer into Normal and Dystrophin-Deficient Mouse Myoblasts 总被引:1,自引:1,他引:0
Abstract: A range of tissue types has now been targeted for development of gene therapeutic procedures both to correct genetic defects and to treat acquired disease. In particular, skeletal muscle holds great importance, not exclusively for the treatment of inherited muscle disorders but also as a platform for the expression of heterologous recombinant proteins, destined to immunise the host or to serve some systemic therapeutic goal. With respect to the X-linked myopathy Duchenne muscular dystrophy (DMD), several gene therapy protocols are being developed that focus on complementing primary genetic defects in the DMD gene by introducing copies of recombinant gene constructs into muscle cells both ex vivo and in vivo. In the present study the potential use of a range of polycationic liposomes as physical gene delivery systems for skeletal muscle has been examined. Using a LacZ reporter gene under optimised conditions up to 40% transfection efficiencies were obtained with the mouse myoblast cell line C2C12. With primary cultures of normal and dystrophin-deficient mdx mouse muscle, up to 10% transfection efficiency was obtained with reporter gene constructs, and high levels of recombinant human dystrophin expression were observed following transfer of dystrophin cDNA gene constructs. These in vitro studies indicate that cationic liposomes can be used to deliver recombinant genes to muscle cells at high efficiency and form a basis to expand investigations into in vivo expression of recombinant dystrophin protein either by direct intramuscular gene transfer or via implantation of transfected myoblasts. 相似文献
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Karen Guerrero Claire Monge Anna Brückner Ülo Puurand Lumme Kadaja Tuuli Käämbre Enn Seppet Valdur Saks 《Molecular and cellular biochemistry》2010,337(1-2):239-249
We studied possible connections of tubulin, microtubular system, and microtubular network stabilizing STOP protein with mitochondria in rat and mouse cardiac and skeletal muscles by confocal microscopy and oxygraphy. Intracellular localization and content of tubulin was found to be muscle type-specific, with high amounts in oxidative muscles, and much lower in glycolytic skeletal muscle. STOP protein localization and content in muscle cells was also muscle type-specific. In isolated heart mitochondria, addition of 1 μM tubulin heterodimer increased apparent K m for ADP significantly. Dissociation of microtubular system into free tubulin by colchicine treatment only slightly decreased initially high apparent K m for ADP in permeabilized cells, and diffusely distributed free tubulin stayed inside the cells, obviously connected to the intracellular structures. To identify the genes that are specific for oxidative muscle, we developed and applied a method of kindred DNA. The results of sequencing and bioinformatic analysis of isolated cDNA pool common for heart and m. soleus showed that in adult mice the β-tubulin gene is expressed predominantly in oxidative muscle cells. It is concluded that whereas dimeric tubulin may play a significant role in regulation of mitochondrial outer membrane permeability in the cells in vivo, its organization into microtubular network has a minor significance on that process. 相似文献
17.
Dean DA 《American journal of physiology. Cell physiology》2005,289(2):C233-C245
The study of muscle physiology has undergone many changes over the past 25 years and has moved from purely physiological studies to those intimately intertwined with molecular and cell biological questions. To ask these questions, it is necessary to be able to transfer genetic reagents to cells both in culture and, ultimately, in living animals. Over the past 10 years, a number of different chemical and physical approaches have been developed to transfect living skeletal, smooth, and cardiac muscle systems with varying success and efficiency. This review provides a survey of these methods and describes some more recent developments in the field of in vivo gene transfer to these various muscle types. Both gene delivery for overexpression of desired gene products and delivery of nucleic acids for downregulation of specific genes and their products are discussed to aid the physiologist, cell biologist, and molecular biologist in their studies on whole animal biology. electroporation; liposomes; plasmids; transfection; gene expression 相似文献
18.
Muscle tissues can be divided into six classes; smooth, fast skeletal, slow skeletal and cardiac muscle tissues for vertebrates, and striated and smooth muscle tissues for invertebrates. We reconstructed phylogenetic trees of six protein genes that are expressed in muscle tissues and, using a newly developed program, inferred the phylogeny of muscle tissues by superimposition of five of those gene trees. The proteins used are troponin C, myosin essential light chain, myosin regulatory light chain, myosin heavy chain, actin, and muscle regulatory factor (MRF) families. Our results suggest that the emergence of skeletal-cardiac muscle type tissues preceded the vertebrate/arthropod divergence (ca. 700 MYA), while vertebrate smooth muscle seemed to evolve independent of other muscles. In addition, skeletal muscle is not monophyletic, but cardiac and slow skeletal muscles make a cluster. Furthermore, arthropod striated muscle, urochordate smooth muscle, and vertebrate muscles except for smooth muscle share a common ancestor. On the other hand, arthropod nonmuscle and vertebrate smooth muscle and nonmuscle share a common ancestor. 相似文献
19.
Differentially expressed fibroblast growth factors regulate skeletal muscle development through autocrine and paracrine mechanisms 总被引:6,自引:0,他引:6
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《The Journal of cell biology》1996,132(6):1151-1159
Several FGF family members are expressed in skeletal muscle; however, the roles of these factors in skeletal muscle development are unclear. We examined the RNA expression, protein levels, and biological activities of the FGF family in the MM14 mouse skeletal muscle cell line. Proliferating skeletal muscle cells express FGF-1, FGF-2, FGF-6, and FGF-7 mRNA. Differentiated myofibers express FGF-5, FGF-7, and reduced levels of FGF-6 mRNA. FGF-3, FGF-4, and FGF-8 were not detectable by RT-PCR in either proliferating or differentiated skeletal muscle cells. FGF-I and FGF-2 proteins were present in proliferating skeletal muscle cells, but undetectable after terminal differentiation. We show that transfection of expression constructs encoding FGF-1 or FGF-2 mimics the effects of exogenously applied FGFs, inhibiting skeletal muscle cell differentiation and stimulating DNA synthesis. These effects require activation of an FGF tyrosine kinase receptor as they are blocked by transfection of a dominant negative mutant FGF receptor. Transient transfection of cells with FGF-1 or FGF-2 expression constructs exerted a global effect on myoblast DNA synthesis, as greater than 50% of the nontransfected cells responded by initiating DNA synthesis. The global effect of cultures transfected with FGF-2 expression vectors was blocked by an anti-FGF-2 monoclonal antibody, suggesting that FGF-2 was exported from the transfected cells. Despite the fact that both FGF-l and FGF-2 lack secretory signal sequences, when expressed intracellularly, they regulate skeletal muscle development. Thus, production of FGF-1 and FGF-2 by skeletal muscle cells may act as a paracrine and autocrine regulator of skeletal muscle development in vivo. 相似文献
20.
Striated muscles, cardiac and skeletal muscles, use calcium as a second messenger to respond and adapt to environmental stimuli. Elevations in intracellular calcium activate calcineurin, a serine/threonine phosphatase, resulting in expression of a set of genes involved in remodeling striated muscle. Activation of calcineurin in hearts produces cardiac hypertrophy, and in skeletal muscle promotes cell differentiation and transforms fiber type specificity. In this review we discuss the effects of calcineurin activity on development, adaptation, and disease of striated muscle. 相似文献