Stable, conditional, and muscle‐fiber‐specific expression of electroporated transgenes in chick limb muscle cells

Wang, Hui; Bonnet, Aline; Delfini, Marie-Claire; Kawakami, Koichi; Takahashi, Yoshiko; Duprez, Delphine

doi:10.1002/dvdy.22498

Cited by 15 publications

(23 citation statements)

References 35 publications

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“…S2A-C, data not shown). In the case of the neural crest (Betancur et al, 2010;Rinon et al, 2011) and dermomyotome (Scaal et al, 2004;Gros et al, 2009;Wang et al, 2011), electroporation appears to result in a greater number of cells being transfected compared to our liposomes (Supp. Fig.…”

Section: Adenovirusmentioning

confidence: 96%

“…The most promising means to integrate foreign DNA without using viruses is the use of transposable elements. Co-electroporation of a plasmid expressing the transposase enzyme with another plasmid expressing a gene of interest (flanked by appropriate recombination sites) results in long-term expression (Sato et al, 2007;Takahashi et al, 2008;Wang et al, 2011). The transposable element approach should also work with lipoplexes.…”

Section: Adenovirusmentioning

confidence: 99%

“…Our method is compatible with a wide range of embryo stages and so could be used to study other developmental processes. In addition, stable integration is possible using the transposase system, which should permit long-term fate-mapping studies (Sato et al, 2007) or cell behavior studies (Gros et al, 2010;Wang et al, 2011). The other clear application is that easy transfer of multiple types of nucleic acid (plasmids, siRNAs, Morpholinos) into cells is possible.…”

Section: Developmental Dynamicsmentioning

confidence: 99%

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Development of high-concentration lipoplexes for in vivo gene function studies in vertebrate embryos

et al. 2011

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Here we report that highly concentrated cationic lipid/helper lipid‐nucleic acid complexes (lipoplexes) can facilitate reproducible delivery of a variety of oligonucleotides and plasmids to chicken embryos or to mouse embryonic mesenchyme. Specifically, liposomes composed of N,N‐dioleyl‐N,N‐dimethylammonium chloride (DODAC)/1,2 dioleoyl glycero‐3‐phosphorylethanolamine (DOPE) prepared at 18‐mM concentrations produced high levels of transfection of exogenous genes in vivo and in vitro. Furthermore, we report sufficient uptake of plasmids expressing interference RNA to decrease expression of both exogenous and endogenous genes. The simplicity of preparation, implementation, and relatively low toxicity of this transfection reagent make it an attractive alternative for developmental studies in post‐gastrulation vertebrate embryos. Developmental Dynamics 240:2108–2119, 2011. © 2011 Wiley‐Liss, Inc.

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Section: Adenovirusmentioning

confidence: 96%

Section: Adenovirusmentioning

confidence: 99%

Section: Developmental Dynamicsmentioning

confidence: 99%

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Development of high-concentration lipoplexes for in vivo gene function studies in vertebrate embryos

et al. 2011

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“…We used somite electroporation in chick embryos (Wang et al, 2011) to assess the effect of mSim2 overexpression on cMyoD expression. Interlimb somites were electroporated in HH16 chick embryos (27 somite stage) with mSim2 and GFP expression vectors or with a GFP expression vector only, as control.…”

Section: Research Article Sim2 and Limb Myogenesismentioning

confidence: 99%

Sim2 prevents entry into the myogenic program by repressing MyoD transcription during limb embryonic myogenesis

et al. 2012

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SUMMARYThe basic helix-loop-helix transcription factor MyoD is a central actor that triggers the skeletal myogenic program. Cellautonomous and non-cell-autonomous regulatory pathways must tightly control MyoD expression to ensure correct initiation of the muscle program at different places in the embryo and at different developmental times. In the present study, we have addressed the involvement of Sim2 (single-minded 2) in limb embryonic myogenesis. Sim2 is a bHLH-PAS transcription factor that inhibits transcription by active repression and displays enhanced expression in ventral limb muscle masses during chick and mouse embryonic myogenesis. We have demonstrated that Sim2 is expressed in muscle progenitors that have not entered the myogenic program, in different experimental conditions. MyoD expression is transiently upregulated in limb muscle masses of Sim2 -/-mice.Conversely, Sim2 gain-of-function experiments in chick and Xenopus embryos showed that Sim2 represses MyoD expression. In addition, we show that Sim2 represses the activity of the mouse MyoD promoter in primary myoblasts and is recruited to the MyoD core enhancer in embryonic mouse limbs. Sim2 expression is non-autonomously and negatively regulated by the dorsalising factor Lmx1b. We propose that Sim2 represses MyoD transcription in limb muscle masses, through Sim2 recruitment to the MyoD core enhancer, in order to prevent premature entry into the myogenic program. This MyoD repression is predominant in ventral limb regions and is likely to contribute to the differential increase of the global mass of ventral muscles versus dorsal muscles.

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“…However, most studies are based on electroporation with the use of transient vectors. Due to the episomal expression of these vectors, the electroporated cells failed to maintain the transgene expression more than 48 to 72 h after electroporation [ 42 – 44 ]. It therefore prevented any study at late stages of development.…”

Section: Introductionmentioning

confidence: 99%

Stable and bicistronic expression of two genes in somite- and lateral plate-derived tissues to study chick limb development

Bourgeois

Lima

Charvet³

et al. 2015

BMC Dev Biol

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BackgroundComponents of the limb musculoskeletal system have distinct mesoderm origins. Limb skeletal muscles originate from somites, while the skeleton and attachments (tendons and connective tissues) derive from limb lateral plate. Despite distinct mesoderm origins, the development of muscle, skeleton and attachments is highly coordinated both spatially and temporally to ensure complete function of the musculoskeletal system. A system to study molecular interactions between somitic-derived tissues (muscles) and lateral-plate-derived tissues (skeletal components and attachments) during limb development is missing.ResultsWe designed a gene delivery system in chick embryos with the ultimate aim to study the interactions between the components of the musculoskeletal system during limb development. We combined the Tol2 genomic integration system with the viral T2A system and developed new vectors that lead to stable and bicistronic expression of two proteins at comparable levels in chick cells. Combined with limb somite and lateral plate electroporation techniques, two fluorescent reporter proteins were co-expressed in stoichiometric proportion in the muscle lineage (somitic-derived) or in skeleton and their attachments (lateral-plate-derived). In addition, we designed three vectors with different promoters to target muscle cells at different steps of the differentiation process.ConclusionLimb somite electroporation technique using vectors containing these different promoters allowed us to target all myogenic cells, myoblasts or differentiated muscle cells. These stable and promoter-specific vectors lead to bicistronic expression either in somitic-derived myogenic cells or lateral plate-derived cells, depending on the electroporation sites and open new avenues to study the interactions between myogenic cells and tendon or connective tissue cells during limb development.

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Stable, conditional, and muscle‐fiber‐specific expression of electroporated transgenes in chick limb muscle cells

Cited by 15 publications

References 35 publications

Development of high-concentration lipoplexes for in vivo gene function studies in vertebrate embryos

Development of high-concentration lipoplexes for in vivo gene function studies in vertebrate embryos

Sim2 prevents entry into the myogenic program by repressing MyoD transcription during limb embryonic myogenesis

Stable and bicistronic expression of two genes in somite- and lateral plate-derived tissues to study chick limb development

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