High-efficiency gene transfer into skeletal muscle mediated by electric pulses.
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Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. However, present DNA delivery technologies have to be improved with regard to both the level of expression and interindividual variability. We report very efficient plasmid DNA transfer in muscle fibers by using square-wave electric pulses of low field strength (less than 300 V/cm) and of long duration (more than 1 ms). Contrary to the electropermeabilization-induced uptake of small molecules into muscle fibers, plasmid DNA has to be present in the tissue during the electric pulses, suggesting a direct effect of the electric field on DNA during electrotransfer. This i.m. electrotransfer method increases reporter and therapeutic gene expression by several orders of magnitude in various muscles in mouse, rat, rabbit, and monkey. Moreover, i.m. electrotransfer strongly decreases variability. Stability of expression was observed for at least 9 months. With a pCMV-FGF1 plasmid coding for fibroblast growth factor 1, this protein was immunodetected in the majority of muscle fibers subjected to the electric pulses. DNA electrotransfer in muscle may have broad applications in gene therapy and in physiological, pharmacological, and developmental studies. (+info)
Escherichia coli genes regulated by cell-to-cell signaling.
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Utilizing the bicistronic reporter transposon mini-Tn5 lacZ-tet/1, we have identified lacZ fusions to four Escherichia coli genes/operons that are strongly activated by the accumulation of self-produced extracellular signals. These fusions were designated cma9, cma48, cma113, and cma114 for conditioned medium activated. Each of the cma fusions was expressed in a growth phase-dependent manner, and the presence of conditioned medium from a stationary phase E. coli culture resulted in the premature activation of these fusions in cells at early to mid-logarithmic phase. The cma48 and cma114 fusions were dependent on RpoS for growth phase expression and response to extracellular factors. The extracellular factors that activated the cma9, cma48, and cma114 fusions were produced in both rich complex and defined minimal media. The cma fusions were shown to be within the cysK (cma9), astD (cma48), tnaB (cma113), and gabT (cma114) genes. These genes function in the uptake, synthesis, or degradation of amino acids that yield pyruvate and succinate. (+info)
Transduction of Aedes aegypti mosquitoes with vectors derived from Aedes densovirus.
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Aedes densovirus (AeDNV)-based constructs that express green fluorescent protein (GFP) from either the P7 or the P61 promoter were made. The construct in which GFP protein was expressed as a fusion protein to the C-terminus of NS1 (NS1-GFP) showed the highest level of GFP expression. This hybrid NS1-GFP protein preserved the biological functions of the parental proteins: it showed GFP fluorescence, it stimulated expression from the virus promoters, and it facilitated rescue and replication of the cloned AeDNV genome. Similar to NS1, the hybrid NS1-GFP localized in the nucleus predominantly in a punctate pattern. Transducing virus particles carrying the NS1-GFP gene infected mosquito larvae. Expression of GFP was detected as early as 48 h postinfection and in larval and pupal stages. Midgut, hindgut, and Malpighian tubule cells expressed GFP soon after transduction. However, the anal papillae were the most commonly infected organ system. The anal papillae are syncytia and regulate ion concentration in the hemolymph of mosquito larvae, and they might be a novel route of mosquito larvae infection with densoviruses. (+info)
Cellular uptake of cationic lipid/DNA complexes by cultured myoblasts and myotubes.
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Several cationic lipids which are highly efficient for delivering genes in vitro do not increase gene delivery in vivo after an intramuscular injection. In order to elucidate the origin of this phenomenon, we have studied the cellular uptake and intracellular fate of cationic lipid/DNA complexes in vitro on myogenic mouse cells (myoblasts and myotubes) of the C2 cell line and of primary cultures. We used a cationic lipid with a spermine head group and its fluorescent analog, and a fluorescent plasmid obtained by nick-translation. In myoblasts, transgene expression was obtained and lipoplexes were internalized in cytoplasmic vesicles. In myotubes, no transgene expression could be detected and we observed an absence of lipoplex internalization. The in vitro uptake of cationic lipid was inversely correlated with the degree of fusion of C2 cell myotubes cultures. (+info)
Structural comparisons of TIM barrel proteins suggest functional and evolutionary relationships between beta-galactosidase and other glycohydrolases.
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Beta-galactosidase (lacZ) from Escherichia coli is a 464 kDa homotetramer. Each subunit consists of five domains, the third being an alpha/beta barrel that contains most of the active site residues. A comparison is made between each of the domains and a large set of proteins representative of all structures from the protein data bank. Many structures include an alpha/beta barrel. Those that are most similar to the alpha/beta barrel of E. coli beta-galactosidase have similar catalytic residues and belong to the so-called "4/7 superfamily" of glycosyl hydrolases. The structure comparison suggests that beta-amylase should also be included in this family. Of three structure comparison methods tested, the "ProSup" procedure of Zu-Kang and Sippl and the "Superimpose" procedure of Diederichs were slightly superior in discriminating the members of this superfamily, although all procedures were very powerful in identifying related protein structures. Domains 1, 2, and 4 of E. coli beta-galactosidase have topologies related to "jelly-roll barrels" and "immunoglobulin constant" domains. This fold also occurs in the cellulose binding domains (CBDs) of a number of glycosyl hydrolases. The fold of domain 1 of E. coli beta-galactosidase is closely related to some CBDs, and the domain contributes to substrate binding, but in a manner unrelated to cellulose binding by the CBDs. This is typical of domains 1, 2, 4, and 5, which appear to have been recruited to play roles in beta-galactosidase that are unrelated to the functions that such domains provide in other contexts. It is proposed that beta-galactosidase arose from a prototypical single domain alpha/beta barrel with an extended active site cleft. The subsequent incorporation of elements from other domains could then have reduced the size of the active site from a cleft to a pocket to better hydrolyze the disaccharide lactose and, at the same time, to facilitate the production of inducer, allolactose. (+info)
Murine tongue muscle displays a distinct developmental profile of MRF and contractile gene expression.
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Few studies have addressed the molecular differences that exist between muscles of the body and those of the craniofacial apparatus. In this study, we characterize the molecular events associated with determination and differentiation of the tongue musculature. We assess the expression of myogenic regulatory factors as well as the developmentally regulated myosin heavy chain, (MHC), genes which serve as markers of differentiation. These results suggest that tongue and limb muscle form by distinct molecular pathways. The myoblasts that contribute to the formation of the tongue preferentially express Myf-5 during myoblast determination rather than MyoD. Subsequently, isolated regions of myogenin expression mark the differentiation of first, the small primary myofibers and later, the larger secondary myofibers. Analysis of differentiation markers demonstrates that the tongue muscle also assumes a unique profile of MHC expression as compared to that of the muscles of the body. Unlike the myoblasts of the developing limb, which express embryonic and neonatal forms of MHC and later express MHC-slow, the tongue myoblasts co-express MHC-embryonic, MHC-slow and MHC-fast isoforms from gestational age E12. Proteins for MHC embryonic and MHC fast isoforms are detected almost simultaneously. Interestingly, MHC-slow transcripts do not appear to be translated into a detectable MHC slow protein at any developmental stage assayed. These results provide further evidence to suggest that skeletal tongue muscle represents a myoblast lineage that develops differently than the limb. (+info)
Transgenic mice ubiquitously expressing human placental alkaline phosphatase (PLAP): an additional reporter gene for use in tandem with beta-galactosidase (lacZ).
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A fundamental keystone of developmental biology has been the growing use of reporter genes in model transgenic systems. Their use has greatly facilitated investigations of cell lineage and cell fate in addition to aiding experiments aimed at determining patterns of gene expression, gene interaction and gene regulation. Through construction of transgenic mice, ubiquitously expressing human placental alkaline phosphatase (PLAP), we demonstrate the suitability of PLAP as a reporter gene for use in conjunction with, or as an alternative to, beta-galactosidase (lacZ). Our findings demonstrate that over-expression of PLAP has no adverse effects on mouse development or viability, despite a widespread pattern of expression. This technology provides a simple yet effective mechanism based on eukaryotic reporter gene technology to facilitate the identification of transgenic cells within complex in vivo systems. (+info)
Improved reporter strain for monitoring Cre recombinase-mediated DNA excisions in mice.
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Effective use of conditional Cre recombinase-loxP gene modification requires Cre-expressing mouse strains with defined patterns of expression. To assess the in vivo functionality of Cre-expressing mice, we have engineered an improved reporter strain for monitoring Cre-mediated excisions. The beta-galactosidase-neomycin phosphotransferase fusion gene (betageo)-trapped ROSA26 locus was modified by gene targeting such that betageo is expressed only after Cre-mediated excision of loxP-flanked DNA sequences. betageo from the excised ROSA26 allele is expressed ubiquitously in embryos and adult mice. By mating the reporter strain with Cre-expressing transgenic mice, we have shown that the loxP-flanked ROSA26 allele is accessible to Cre during early embryogenesis, as well as in a specific hematopoietic lineage (T lymphocytes). This improved reporter strain should facilitate monitoring in vivo Cre-mediated excision events in a variety of experimental contexts. (+info)