title: Stable and bicistronic expression of two genes in somite- and lateral plate-derived tissues to study chick limb development.
abstract: BACKGROUND
  Components 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  Limb 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.
authors.affiliation: CNRS UMR 7622, IBPS-Developmental Biology Laboratory, F-75005, Paris, France. la_logique@hotmail.com.|Sorbonne Universités, UPMC Univ Paris 06, IBPS-Developmental Biology Laboratory, F-75005, Paris, France. la_logique@hotmail.com.|Inserm U1156, F-75005, Paris, France. la_logique@hotmail.com.
authors.forename: Adeline
authors.initials: A
authors.lastname: Bourgeois
authors.affiliation: CNRS UMR 7622, IBPS-Developmental Biology Laboratory, F-75005, Paris, France. joana.lima@upmc.fr.|Sorbonne Universités, UPMC Univ Paris 06, IBPS-Developmental Biology Laboratory, F-75005, Paris, France. joana.lima@upmc.fr.|Inserm U1156, F-75005, Paris, France. joana.lima@upmc.fr.
authors.forename: Joana
authors.initials: J
authors.lastname: Esteves de Lima
authors.affiliation: CNRS UMR 7622, IBPS-Developmental Biology Laboratory, F-75005, Paris, France. charvet.benjamin@voila.fr.|Sorbonne Universités, UPMC Univ Paris 06, IBPS-Developmental Biology Laboratory, F-75005, Paris, France. charvet.benjamin@voila.fr.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Charvet
authors.affiliation: Division of Molecular and Developmental Biology, National Institute of Genetics, and Department of Genetics, SOKENDAI (The Graduate University for Advanced Studies), Mishima, Shizuoka, Japan. kokawaka@nig.ac.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Kawakami
authors.affiliation: Institue for Chemistry and Biochemistry, Freie Universitaet Berlin, 14195, Berlin, Germany. sigmar.stricker@fu-berlin.de.
authors.forename: Sigmar
authors.initials: S
authors.lastname: Stricker
authors.affiliation: CNRS UMR 7622, IBPS-Developmental Biology Laboratory, F-75005, Paris, France. delphine.duprez@upmc.fr.|Sorbonne Universités, UPMC Univ Paris 06, IBPS-Developmental Biology Laboratory, F-75005, Paris, France. delphine.duprez@upmc.fr.|Inserm U1156, F-75005, Paris, France. delphine.duprez@upmc.fr.
authors.forename: Delphine
authors.initials: D
authors.lastname: Duprez
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D001842:Bone and Bones / Q000196:embryology*; D002642:Chick Embryo; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000235:genetics; D018274:Electroporation; D005121:Extremities / Q000196:embryology*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D018878:Limb Buds / Q000196:embryology*; D024510:Muscle Development / Q000502:physiology; D018482:Muscle, Skeletal / Q000196:embryology*; D018994:Myosin Light Chains / Q000235:genetics; D038081:Organogenesis / Q000235:genetics / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics; D019170:Somites / Q000196:embryology*
chemical_list: D000199:Actins; D050761:Cyclin-Dependent Kinase Inhibitor p57; D018994:Myosin Light Chains; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins