title: Expression of fluorescent proteins in Branchiostoma lanceolatum by mRNA injection into unfertilized oocytes.
abstract: We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.
authors: Département de Biologie du Développement et Cellules Souches, Institut Pasteur.
authors: Estelle
authors: E
authors: Hirsinger
authors: Laboratoire de Biologie du Développement de Villefranche-sur-Mer (UMR7009 CNRS/UPMC Univ Paris 06), Sorbonne Universités.
authors: João Emanuel
authors: JE
authors: Carvalho
authors: Département de Biologie du Développement et Cellules Souches, Institut Pasteur; Equipe Epigenetic Control of Normal and Pathological Hematopoiesis, Centre de Recherche en Cancérologie de Marseille.
authors: Christine
authors: C
authors: Chevalier
authors: Unité de Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS UMR5235/DAA/cc107/Université Montpellier II.
authors: Georges
authors: G
authors: Lutfalla
authors: Département de Biologie du Développement et Cellules Souches, Institut Pasteur.
authors: Jean-François
authors: JF
authors: Nicolas
authors: Plateforme BioEmergences IBiSA FBI, CNRS-NED, Institut de Neurobiologie Alfred Fessard; nadine.peyrieras@inaf.cnrs-gif.fr.
authors: Nadine
authors: N
authors: Peyriéras
authors: Laboratoire de Biologie du Développement de Villefranche-sur-Mer (UMR7009 CNRS/UPMC Univ Paris 06), Sorbonne Universités.
authors: Michael
authors: M
authors: Schubert
mesh_terms: D000818:Animals; D005260:Female; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D064175:Lancelets / Q000378:metabolism*; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D008297:Male; D008845:Microinjections / Q000379:methods*; D009865:Oocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012333:RNA, Messenger; D000097573:Red Fluorescent Protein
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