Nous sommes reconnaissants à Nancy Papalopulu et Boyan Bonev de l&#39;aide pour l&#39;électroporation Xenopus. Nous remercions également Marc Dionne pour la lecture critique, Jeremy Green et John Wallingford pour des discussions utiles et des membres du laboratoire Liu pour leur soutien. Ce travail a été financé par des subventions du BBSRC (BB/E013872/1) et le Wellcome Trust (081880/Z/06/Z) pour KJL.

Partie 1A. Équipement Microscope: debout champ stéréoscopique avec l&#39;objectif de faible puissance <ul><li> Tension / Générateur d&#39;impulsions: BTX ECM 830 Système Place électroporation vague </li><li> Pipeter extracteur: P-87 Micropipette Puller (Sutter Instrument Company, CA) </li><li> Manipulateur: grossier, ou combinés grossier et fin en fonction de la préparation. </li><li> Micropipette titulaire: Outils Fine Science </li><li> Électrode: maison </li><li> L&#39;électroporation de chambre: maison </li></ul> L-électrodes en forme: <ol><li> Couper 8 cm de haute pureté 0,4 mm fil de tungstène (Goodfellow) et apposer au milieu d&#39;une seringue de 1 ml d&#39;utiliser du mastic (nous utilisons la Blu-Tack). Laisser 4 cm de fil de tungstène exposée de la pointe de la seringue et l&#39;embout plier en forme de L, 1 cm de l&#39;extrémité (fig. 1A). </li><li> Garniture pointe de sorte que la fin des mesures de 0,5 mm de longueur. Tson pourboire est le terminus de l&#39;électrode. </li><li> Exécutez l&#39;excès parallèles fil de tungstène à la seringue et l&#39;utiliser pour connecter le générateur d&#39;impulsions de l&#39;électrode. </li><li> Répétez le processus faisant une paire d&#39;électrodes. </li><li> Attacher les électrodes au générateur d&#39;ondes carrées d&#39;impulsion via des câbles à courant continu. </li></ol> L&#39;électroporation de chambre <ol><li> Tapisser le fond plat de 90 mm avec ~ 5 mm modeler non toxique. </li><li> Remplissez plat avec les médias électroporation. </li><li> En utilisant une pince n ° 5 horloger se tailler un puits en forme de T (fig. 2). Le bien longtemps devrait mesurer environ 2 mm x 2 mm x 10 mm et le court ~ 2 mm x 2 mm x 5 mm. Le plat électroporation peuvent être lavés et réutilisés. </li></ol> Partie 1B. Réactifs ADN ou macromolécules chargées <ul><li> Micropipettes: 1 mm de large 4 &quot;de long en verre borosilicate capillaires (WPI, TW100-F) </li><li> Milieux de culture: Normal amphibiens médias (NAM) </li><li> &gt; 10 X stock: 1100 mM NaCl, 20mM de KCl, 10 mM de Ca (NO 3) 2 • 4H 2 O, 1 mM EDTA. <ul><li> Autoclave et conserver à 4 ° C. </li><li> 1 X NAM: Diluer de stock, tampon 10x avec 0,1 mM NaHCO 3 et 0,2 mM de Na 3 PO 4. </li></ul></li><li> Têtards Xenopus laevis, le stade 28 </li></ul> Préparation de l&#39;ADN: <ol><li> Préparer des plasmides d&#39;expression utilisant des protocoles standard. </li><li> Resuspendre l&#39;ADN à une concentration finale de 1 ug / ul dans nucléase libre H 2 0. </li></ol> * Nous avons eu du succès avec des vecteurs contenant un promoteur fort CMV, comme pCS2 + [16]. Pour l&#39;analyse de la lignée, nous avons généralement des ADN codant la protéine fluorescente verte (GFP + pCS2) à une concentration finale de 0,1 pg / pl. Les concentrations d&#39;ADN [entre 0,1 à 3 pg / pl ont également été testés. Nous avons constaté que des concentrations inférieures à 0,8 pg / pl cellules inefficace étiquetés, tandis que des concentrations d&#39;ADN de plus de 2 pg / m &amp;u; L n&#39;a pas d&#39;améliorer l&#39;efficacité d&#39;électroporation]. Morpholino oligonucléotides préparation: (Remarque:. OM doivent être fluorescéine (3&#39;-carboxyfluorescéine modifié) ou autrement chargés) <ol><li> Resuspendre oligonucléotides morpholino (OM) (Genetools, <a href="http://www.genetools.com" target="_blank">www.genetools.com</a> ) à une concentration de 2 mM dans nucléase libre H 2 0. </li><li> Aliquote de la chaleur de la solution mère à 65 ° C pendant 5 minutes. </li><li> Diluer à la concentration finale de 0,5 mM dans l&#39;eau sans nucléase. </li></ol> * 0.1-1mm solutions MO ont été testés. 0,5 mM solutions MO ont été suffisantes pour l&#39;électroporation de nombreuses cellules mésenchymateuses. Micropipettes <ol><li> Préparer Micropipettes capillaires à partir de verre borosilicate (1 mm de large, 4 &quot;de long, WPI pas. TW100-F). Utilisez extracteur d&#39;aiguilles pour préparer micropipettes avec un cône 8-12 mm de long et à pointe fine. </li><li> Astuce Crush ~ 2 mmde la pointe avec une pince, en créant une pause déchiquetées. </li></ol> Médias <ol><li> Milieux d&#39;incubation: Préparer une solution fraîche 3 / 4 normal amphibiens médias (MNA) de 1x stock. Ajouter 0,025 mg / ml de gentamycine. </li><li> L&#39;électroporation des médias: comme ci-dessus, avec 0,1% de benzocaïne (Sigma, 06950). </li></ol> 2. L&#39;électroporation Micropipette configuration <ol><li> Remplissez micropipette avec ~ 1 solution injectable ul. </li><li> Fixez micropipette au micromanipulateur et joindre à microinjecteur (Picospritzer II). </li><li> Micropipette angle à 50 ° de la table. </li><li> Régler la pression d&#39;injection à 20 PSI. </li><li> Calibrer micropipette à injecter 30 nl par impulsion. </li></ol> Préparation Tadpole <ol><li> Anesthetize stade de 28 larves de Xenopus par l&#39;incubation dans les médias électroporation pendant 5 minutes. </li><li> Transfert de têtard anesthésiés dans la chambre d&#39;électroporation remplis avec les médias électroporation. Position d&#39;embryondans le temps et afin que la tête repose dans le T-jonction avec la face dorsale vers le bas et la face ventrale exposés. La tête doit être légèrement surélevée par rapport à la queue. </li><li> En utilisant des pinces, doucement sécurisé têtard bien avec entourant la plasticine. (Note:.. Si le têtard n&#39;est pas sécurisé, il peut se contracter et contact des électrodes au cours électroporation Dans ce cas, jetez le têtard que mouchoirs seront sévèrement endommagé) </li></ol> L&#39;électroporation <ol><li> Insérer le bout micropipette immédiatement postérieure à la glande du ciment et dans le mésenchyme du visage. </li><li> Injecter solution à 30 nl en mésenchyme. </li><li> Rétracter micropipette. </li><li> Aligner rapidement des conseils d&#39;électrode parallèle à la tête de l&#39;embryon (fig. 3). </li><li> Appliquer 8 50 ms, impulsions 20mV carrés. </li><li> Rétracter électrodes. </li><li> En utilisant des pinces attentivement communiqué de têtard à partir ainsi et le transfert à 3 / 4 NAM, 0,025 mg / ml de gentamycine. </li><li> Les têtards peuvent être incubés dans 3 / 4 NAM, 0,025 mg / ml overnight, ou plus. </li><li> Embryons d&#39;écran pour l&#39;électroporation efficace par microscopie à fluorescence après 24 heures. </li></ol> 3. Les résultats représentatifs: L&#39;utilisation de molécules fluorescentes permet un dépistage facile d&#39;embryons électroporé. La figure 4 montre un lot typique de MO électroporées têtards ~ 12, 48 et 96 heures après l&#39;électroporation, incubés à 14,5 ° C. En utilisant la microscopie à fluorescence, les OM peuvent être visualisées immédiatement après l&#39;électroporation et persister pendant plusieurs jours après l&#39;électroporation. Dans notre expérience, la fluorescence est faiblement évidente au stade 46 (~ 5 jours plus tard). Dans les cartilages, la fluorescence diminue considérablement après l&#39;apparition de la différenciation (~ st 42); cependant, la fluorescence persiste MO plus fortement dans d&#39;autres types cellulaires tels que l&#39;endoderme du pharynx. La microscopie à fluorescence montre que les oligonucléotides sont incorporées dans plusieurs tissus dont le cartilage craniofaciales. Oligonucléotidiques par fluorescence cune souvent être visualisées dans le tissu de chaque côté de la tête. Cela est probablement dû à la diffusion rapide de la solution injectable dans le mésenchyme lâche craniofaciales avant l&#39;électroporation. <img alt="Figure 1" src="/files/ftp_upload/3381/3381fig1.jpg" /> Figure 1 électrodes maison. Fil de tungstène en forme de L est attachée à une seringue de 1 ml à l&#39;aide non-toxique d&#39;argile ou de mastic. (A) Le terminus d&#39;électrode mesures 5 mm. (B) Joindre une paire d&#39;électrodes, de sorte que les terminus sont parallèles. Des électrodes sont fixées à générateur d&#39;impulsions par des câbles à courant continu. <img alt="Figure 2" src="/files/ftp_upload/3381/3381fig2.jpg" /> Figure 2 chambre d&#39;électroporation. (A) 90 mm plat bordé avec de la plasticine est rempli avec les médias et une chambre en forme de T sculptés avec une pince Aucun horloger de 5. (B) Le côté le plus long mesure 2 mm x 2 mm X10 mmtandis que les mesures à court de 2 mm x 2 mm x 5 mm. Le chef de l&#39;embryon repose dans la jonction en T, la face ventrale en place. <img alt="Figure 3" src="/files/ftp_upload/3381/3381fig3.jpg" /> Figure 3 Procédure électroporation schématique illustrant. St. 28 têtard est placé dans la chambre d&#39;électroporation, la face ventrale en place. Micropipette est inséré dans la glande de ciment mésenchyme facial sous-jacent. Injecter. Micropipette est enlevé et la L-électrodes en forme sont alignés parallèlement flanquant la tête. Appliquer huit de 50 ms, 20 mV impulsions carrées. Rétracter électrodes. Cultivez têtards au stade désiré. Visualisez MOS ou expression de la GFP en utilisant la microscopie à fluorescence. <img alt="Figure 4" src="/files/ftp_upload/3381/3381fig4.jpg" /> Figure 4 têtards représentant 12 (A), 48 (B) et 96 (C) heures après l&#39;électroporation (stades 30, 34 et 44 respectivement). (A &quot;B&quot;) Fluorescent MO peuvent être visualisés au sein de mésenchyme cranio au cerfes 30 et 34. Fluorescence peut être détectée dans les cartilages au stade 44 (pointe de flèche, C&#39;-C &quot;). L&#39;intestin est très autofluorescente.

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Les auteurs n&#39;ont aucun conflit d&#39;intérêts.

Dans cette vidéo, nous avons démontré la faisabilité de la livraison de gènes médiée par électroporation dans le mésenchyme du visage de têtards de Xenopus. En utilisant cette approche, nous pouvons contourner les premiers effets du développement de la manipulation de la fonction des gènes qui nous permet de cibler des tissus spécifiques à des points plus tard. Nos études montrent que les populations hétérogènes de cellules mésenchymateuses craniofaciales peuvent être touchés, ce qui nous permet d&#39;examiner la lignée de cellules par électroporation des cellules ainsi que des exigences autonomes pour des protéines d&#39;intérêt. Combiné avec l&#39;imagerie en direct, nous pouvons utiliser cette approche pour étudier la fonction des gènes, au fil du temps, au cours du développement cranio-facial. Cette nouvelle méthode met en évidence la traçabilité de Xenopus pour l&#39;étude de l&#39;organogenèse. Nous prévoyons que cette méthode peut être largement adapté à l&#39;étude la morphogenèse et la différenciation des tissus d&#39;autres aussi.

A correction was made to <a href="http://http://www.jove.com/video/3381">Electroporation of Craniofacial Mesenchyme</a>. There was an incorrect unit used for the voltage.

Erratum: Electroporation of Craniofacial Mesenchyme

electroporation of craniofacial mesenchyme

L&#39;électroporation est une méthode efficace de délivrer de l&#39;ADN et d&#39;autres macromolécules chargées dans les tissus à des moments précis et dans des endroits précis. Par exemple, l&#39;électroporation a été utilisée avec succès pour étudier le développement neural et rétiniennes chez Xenopus, le poulet et la souris 1-10. Toutefois, il est important de noter que dans toutes ces études, les enquêteurs ne visaient pas les tissus mous. Parce que nous sommes intéressés au développement cranio-faciales, nous avons adapté une méthode pour cibler mésenchyme facial. Lorsque nous avons cherché la littérature, nous avons trouvé, à notre grande surprise, très peu de rapports de transfert de gènes réussie dans le tissu cartilagineux. La majorité de ces études étaient des études de thérapie génique, comme la livraison de siRNA ou de protéines dans des lignées cellulaires chondrogénique, ou des modèles animaux d&#39;arthrite 11-13. Dans d&#39;autres systèmes, comme le poulet ou la souris, l&#39;électroporation de mésenchyme facial a été difficile (COMMUNICAT personnellesions, Département du développement cranio-facial, KCL). Nous avons supposé que l&#39;électroporation dans les tissus cartilagineux et procartilaginous chez Xenopus pourrait mieux fonctionner. Dans nos études, nous montrons que le transfert de gène dans les cartilages faciaux se produit de manière efficace à un stade précoce (28), lorsque l&#39;ébauche du visage est toujours composé de tissus mous avant de différenciation du cartilage. Xenopus est un système très accessible pour l&#39;analyse des vertébrés développement cranio-facial. Structures cranio-faciale sont plus facilement visibles chez Xenopus que dans n&#39;importe quel modèle d&#39;autres vertébrés, principalement parce que les embryons de xénope sont fécondés à l&#39;extérieur, permettant l&#39;analyse des premières étapes, et en facilitant l&#39;imagerie en direct à la résolution seule cellule, ainsi que la réutilisation des 14 mères. Parmi les modèles de développement des vertébrés à l&#39;extérieur, Xenopus est plus utile pour l&#39;analyse craniofaciale que le poisson-zèbre, comme les larves de Xenopus sont plus grandes et plus faciles à dissect, et la région en développement du visage est plus accessible à l&#39;imagerie de la région équivalente dans le poisson. En outre, les Xenopus sont évolutivement plus proche de l&#39;homme que le poisson-zèbre (environ 100 millions ans plus près) 15. Enfin, à ces stades, les têtards de Xenopus sont transparentes, et l&#39;expression simultanée des protéines fluorescentes ou des molécules permettant une visualisation facile des cartilages en développement. Nous prévoyons que cette approche nous permettra de tester rapidement et efficacement des molécules candidates dans un système modèle in vivo.

Watch this Scientific Journal Video about Electroporation of Craniofacial Mesenchyme at JoVE.com

Electroporation of Craniofacial Mesenchyme

Cartilages craniofaciale développent en contact étroit avec d&#39;autres tissus et sont difficiles à manipuler des animaux vivants. Nous utilisons l&#39;électroporation pour offrir des outils moléculaires lors de la croissance du squelette cranio-facial tout en contournant les premiers effets embryonnaire. Cette approche nous permettra de tester efficacement des molécules candidates<em&gt; In vivo.</em

Electroporation de Mesenchyme craniofaciale

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

electroporation-of-craniofacial-mesenchyme

Research

JoVE Journal

Biology

11.8K vues.  King's College London. Cartilages craniofaciale développent en contact étroit avec d'autres tissus et sont difficiles à manipuler des animaux vivants. Nous utilisons l'électroporation pour offrir des outils moléculaires lors de la croissance du squelette cranio-facial tout en contournant les premiers effets embryonnaire. Cette approche nous permettra de tester efficacement des molécules candidates In vivo.

Vidéo: Electroporation of Craniofacial Mesenchyme

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Electroporation de mycobactéries

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

Recherche

Biologie

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

Évaluation des propriétés de signalisation ectodermique épithéliums au cours du développement cranio-facial

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

En électroporation Ovo dans la rétine de poussin embryonnaire

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Un test pour évaluer le comportement des explants cellulaires du mésenchyme crânienne

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

La délétion de gènes dans le génome entier<em&gt; Streptococcus sanguinis</em&gt; Par PCR à haut débit

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

Séparation de souris embryonnaires ectoderme visage et Mesenchyme

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualisation de développement cranio-facial dans les SOX10: Kaede poisson zèbre transgénique ligne à l&#39;aide Time-lapse de microscopie confocale

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analyser craniofaciale morphogenèse chez le poisson zèbre Utilisation de 4D microscopie confocale

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Cultures alternatives pour souches pluripotentes humaines production cellulaire, le maintien et l&#39;analyse génétique

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

Évaluation des contributions spécifiques aux espèces Vers développement cranio-facial Utilisation chimères caille-canard

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...