Wir sind dankbar, dass Nancy Papalopulu und Boyan Bonev für die Unterstützung bei Xenopus Elektroporation. Wir danken auch Marc Dionne für die kritische Durchsicht, Jeremy Green und John Wallingford für hilfreiche Diskussionen und die Mitglieder des Liu-Labor für ihre Unterstützung. Diese Arbeit wurde durch Zuschüsse aus dem BBSRC (BB/E013872/1) und dem Wellcome Trust (081880/Z/06/Z) zu KJL finanziert.

Teil 1A. Ausrüstung Mikroskop: aufrecht Stereo-Binokular mit Low-Power-Ziel <ul><li> Spannung / Pulse Generator: BTX ECM 830 Square Wave Elektroporationssystem </li><li> Pipette puller: P-87 Micropipette Puller (Sutter Instrument Company, CA) </li><li> Manipulator: Grob-oder kombinierte Grob-und Fein je nach Zubereitung. </li><li> Micropipette Inhaber: Fine Science Tools </li><li> Elektrode: hausgemachte </li><li> Elektroporation Kammer: homemade </li></ul> L-förmige Elektroden: <ol><li> Cut 8 cm von hochreinem 0,4 mm Wolframdraht (Goodfellow) und bringen sich in der Mitte ein 1ml Spritze mit putty (wir verwenden Blu-Tack). Leave 4 cm Wolframdraht von der Spitze der Spritze und biegen Spitze in L-Form ausgesetzt, 1 cm vom Ende (Abb. 1A). </li><li> Trim Spitze, so dass am Ende 0,5 mm misst in Länge. TSein Tipp ist die Elektrode Terminus. </li><li> Run über Wolframdraht parallel zur Spritze und es verwenden, um die Elektrode Impulsgeber anschließen. </li><li> Wiederholen Sie den Vorgang macht ein Paar von Elektroden. </li><li> Bringen Elektroden Rechteckimpuls-Generator über DC-Kabel. </li></ol> Elektroporation Kammer <ol><li> Den Boden 90 mm Schale mit ca. 5 mm ungiftige Knete. </li><li> Füllen Gericht mit Elektroporation Medien. </li><li> Mit Nr. 5 Uhrmacher-Pinzette schnitzen eine T-förmige Vertiefung (Abb. 2). Die lange gut gemessen werden sollte ~ 2 mm x 2 mm x 10 mm und die kurzen ~ 2 mm x 2 mm x 5 mm. Die Elektroporation Gericht kann gewaschen und wiederverwendet werden. </li></ol> Teil 1B. Reagenzien DNA oder geladene Makromoleküle <ul><li> Mikropipetten: 1 mm breit 4 &quot;lang Borosilikatglas Kapillaren (WPI, TW100-F) </li><li> Kulturmedien: Normal Amphibian Media (NAM) </li><li> &gt; 10 X Bestand: 1100 mM NaCl, 20mM KCL, 10 mM Ca (NO 3) 2 • 4H 2 O, 1 mM EDTA. <ul><li> Autoklaven und lagern bei 4 ° C. </li><li> 1 X NAM: Verdünnen von 10x Lager, Puffer mit 0,1 mM NaHCO 3 und 0,2 mM Na 3 PO 4. </li></ul></li><li> Xenopus laevis Kaulquappen, Stufe 28 </li></ul> DNA-Präparation: <ol><li> Bereiten Expressionsplasmide über Standardprotokolle. </li><li> Resuspendieren DNA auf eine Endkonzentration von 1 ug / ul in Nuklease freies H 2 0. </li></ol> * Wir hatten Erfolg mit Vektoren, die einen starken CMV-Promotor, wie pCS2 + [16]. Für Abstammung Analyse, wir gehören in der Regel DNA, grün fluoreszierende Protein (GFP pCS2 +) bei einer Endkonzentration von 0,1 ug / ul. [DNA-Konzentrationen zwischen 0,1-3 ug / ul wurden ebenfalls getestet. Wir fanden, dass die Konzentrationen unterhalb von 0,8 ug / ul ineffizient markierten Zellen, während DNA-Konzentrationen von mehr als 2 g / m &amp;u; l nicht verbessert Elektroporation Effizienz]. Morpholino Oligonukleotid Zubereitung: (Hinweis:. MOs müssen fluoresceinated werden (3&#39;-Carboxyfluorescein modifiziert) oder anderweitig gegen Gebühr) <ol><li> Resuspendieren Morpholino-Oligonukleotide (MOs) (GeneTools, <a href="http://www.genetools.com" target="_blank">www.genetools.com</a> ) in einer Konzentration von 2 mM in Nuklease freies H 2 0. </li><li> Hitze Aliquot der Stammlösung bei 65 ° C für 5 Minuten. </li><li> Verdünnen, um eine Endkonzentration von 0,5 mM in Nuclease freies Wasser. </li></ol> * 0,1-1mM MO-Lösungen getestet. 0,5 mM MO-Lösungen reichten für die Elektroporation von vielen mesenchymalen Zellen. Mikropipetten <ol><li> Bereiten Mikropipetten aus Borosilikatglas Kapillaren (1 mm breit, 4 &quot;lang, WPI-Nr. TW100-F). Verwenden Nadel puller zu Mikropipetten mit einem 8-12 mm langen Kegel und feine Spitze vorzubereiten. </li><li> Crush-Spitze ~ 2 mmVon der Spitze mit einer Pinzette, die Schaffung eines zerklüfteten brechen. </li></ol> Medien <ol><li> Incubation Medien: Bereiten Sie frischen 3 / 4 Normal-Amphibian Media (NAM) von 1x Lager. Add 0,025 mg / ml Gentamycin. </li><li> Elektroporation Medien: wie oben, mit 0,1% Benzocain (Sigma, 06950). </li></ol> 2. Elektroporation Micropipette Setup <ol><li> Füllen Mikropipette mit ~ 1 ul Injektionslösung. </li><li> Sichere Mikropipette in Mikromanipulator und heften sich an Mikroinjektor (Picospritzer II). </li><li> Angle Mikropipette bei 50 ° von der Tischplatte. </li><li> Set Einspritzdruck bei 20 PSI. </li><li> Kalibrieren Mikropipette zu 30 nl pro Puls zu injizieren. </li></ol> Tadpole Vorbereitung <ol><li> Anesthetize Stufe 28 Xenopus Larven durch Inkubation in Elektroporation Medien für 5 Minuten. </li><li> Transfer narkotisierten Kaulquappe in Elektroporation Kammer mit Elektroporation Medien gefüllt. Position Embryoinnerhalb der langen gut, so dass der Kopf in der T-Kreuzung mit dorsalen Seite nach unten und Bauchseite ausgesetzt ruht. Der Kopf sollte leicht im Vergleich zu den Schwanz angehoben werden. </li><li> Mit einer Pinzette vorsichtig zu sichern Kaulquappe in gut mit den umliegenden Plastilin. (Hinweis:.. Wenn die Kaulquappen nicht gesichert ist, kann es zucken und Kontaktelektrode bei der Elektroporation In diesem Fall verwerfen die Kaulquappe als Kosmetiktücher schwer beschädigt wird) </li></ol> Elektroporation <ol><li> Legen Sie Mikropipette Spitze unmittelbar hinter der Zement-Drüse und in Gesichts-Mesenchym. </li><li> Inject 30 nl Lösung in Mesenchym. </li><li> Retract Mikropipette. </li><li> Schnell align Elektrodenspitzen parallel zum Kopf des Embryos (Abb. 3). </li><li> Bewerben 8 50 ms, 20mV Rechteckimpulse. </li><li> Retract Elektroden. </li><li> Mit einer Pinzette vorsichtig Entlassung aus gut Kaulquappe und Transfer zum 3 / 4 NAM, 0,025 mg / ml Gentamycin. </li><li> Kaulquappen können in 3 / 4 NAM, 0,025 mg / ml Übernacht inkubiert werdenight, oder länger. </li><li> Screen-Embryonen für die effiziente Elektroporation durch Fluoreszenzmikroskopie nach 24 Stunden. </li></ol> 3. Repräsentative Ergebnisse: Die Verwendung von fluoreszierenden Molekülen erlaubt eine einfache Screening von Embryonen elektroporiert. Abbildung 4 zeigt eine typische Charge von MO elektroporiert Kaulquappen ~ 12, 48 und 96 Stunden nach der Elektroporation mit 14,5 ° C inkubiert Mit Fluoreszenz-Mikroskopie kann MOs unmittelbar nach der Elektroporation visualisiert werden und bleiben für einige Tage nach der Elektroporation. Nach unserer Erfahrung ist die Fluoreszenz schwach erkennbar im Stadium 46 (ca. 5 Tage später). In den Knorpeln, sinkt dramatisch Fluoreszenz nach dem Beginn der Differenzierung (~ st 42), jedoch besteht MO Fluoreszenz stärker in andere Zelltypen wie der pharyngealen Endoderm. Die Fluoreszenzmikroskopie zeigt, dass Oligonukleotide in mehreren kraniofazialen Gewebe einschließlich Knorpel eingebaut werden. Oligonukleotid-Fluoreszenz ceine oft im Gewebe auf beiden Seiten des Kopfes sichtbar gemacht werden. Dies ist wahrscheinlich auf eine schnelle Diffusion der Injektionslösung während der lose kraniofazialen Mesenchym vor der Elektroporation. <img alt="Abbildung 1" src="/files/ftp_upload/3381/3381fig1.jpg" /> Abbildung 1 Homemade Elektroden. L-förmige Wolframdraht ist es, eine 1 ml Spritze mit nicht-toxischen oder Kitt befestigt. (A) Die Elektrode terminus misst 5 mm. (B) Befestigen Sie ein Paar von Elektroden, so dass die Enden parallel laufen. Die Elektroden sind an Impulsgeber von DC-Kabel angeschlossen. <img alt="Abbildung 2" src="/files/ftp_upload/3381/3381fig2.jpg" /> Abbildung 2 Elektroporation Kammer. (A) 90 mm-Schale mit Plastilin gesäumt ist mit Medien und eine T-förmige Kammer mit Nr. 5 Uhrmacher-Pinzette geschnitzt gefüllt. (B) Die lange Seite misst 2 mm X 2 mm X10 mmwährend der kurzen misst 2 mm X 2 mm X 5 mm. Der Kopf des Embryos liegt in der T-Kreuzung, Bauchseite auf. <img alt="Abbildung 3" src="/files/ftp_upload/3381/3381fig3.jpg" /> Abbildung 3 Schematische Darstellung der Elektroporation Verfahren. St. 28 Kaulquappe ist in Elektroporation Kammer platziert, Bauchseite auf. Mikropipette in Gesichts-Mesenchym zugrunde liegenden Zement Drüse eingefügt. Inject. Mikropipette wird entfernt und L-förmigen Elektroden sind parallel ausgerichtet flankierenden den Kopf. Wenden Sie acht 50 ms, 20 mV Rechteckimpulse. Retract Elektroden. Wachsen Kaulquappen zu wünschen Stufen. Visualisieren MOs oder GFP-Expression mittels Fluoreszenzmikroskopie. <img alt="Abbildung 4" src="/files/ftp_upload/3381/3381fig4.jpg" /> Abbildung 4 Vertreter Kaulquappen 12 (A), 48 (B) und 96 (C) Stunden nach der Elektroporation (Stufen 30, 34 bzw. 44). (A &quot;-B&quot;) Fluorescent MO innerhalb kraniofazialen Mesenchym bei Hirsch visualisiert werdenES 30 und 34 Jahren. Fluoreszenz kann in Knorpel im Stadium 44 (Pfeilspitze, C&#39;-C &quot;) nachgewiesen werden. Der Darm ist sehr autofluoreszierenden.

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Die Autoren haben keinen Interessenkonflikt.

In diesem Video haben wir die Machbarkeit der Elektroporation-vermittelte Gentransfer in die Gesichts-Mesenchym von Xenopus Kaulquappen nachgewiesen. Mit diesem Ansatz können wir umgehen frühen Entwicklungsstadium Einfluss der Manipulation von Gen-Funktion ermöglicht es uns, spezifische Gewebe zu späteren Zeitpunkten Ziel. Unsere Studien zeigen, dass heterogene Populationen von kraniofazialen mesenchymalen Zellen beeinflusst werden kann, so dass wir Abstammung elektroporiert Zellen sowie Zell-autonome Anforderungen für Proteine ​​von Interesse zu untersuchen. In Kombination mit Live-Bildgebung können wir mit diesem Ansatz Genfunktion Studie über die Zeit, während der kraniofazialen Entwicklung. Dieses neuartige Verfahren hebt die Lenkbarkeit des Xenopus für das Studium der Organogenese. Wir gehen davon aus, dass diese Methode im Großen und Ganzen kann angepasst werden, um Morphogenese und Differenzierung von anderen Geweben sowie zu studieren.

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

Elektroporation ist eine effiziente Methode der Bereitstellung von DNA und andere geladene Makromoleküle in das Gewebe zu präzisen Zeitpunkten und in genauen Standorte. Zum Beispiel, Elektroporation wurde mit großem Erfolg verwendet worden, um Nerven-und Retina-Entwicklung in Xenopus-Studie, Huhn und Maus 1-10. Es ist jedoch wichtig anzumerken, dass in all diesen Studien, Ermittler wurden nicht gezielt Weichteile. Weil wir in kraniofazialen Entwicklung interessiert sind, passten wir eine Methode zur Gesichts-Mesenchym Ziel. Wenn wir die Literatur gesucht, fanden wir zu unserer Überraschung, nur sehr wenige Berichte über erfolgreiche Gentransfer in Knorpelgewebe. Die meisten dieser Studien waren Gentherapie-Studien, wie siRNA oder Proteinen Lieferung in chondrogene Zelllinien, oder Tiermodellen von Arthritis 11-13. In anderen Systemen, wie Huhn oder eine Maus, die Elektroporation von Gesichts-Mesenchym war eine echte Herausforderung (persönliche KommunikIonen, Dept of Craniofacial Development, KCL). Wir stellten die Hypothese, dass Elektroporation in procartilaginous und knorpeligen Gewebe in Xenopus könnte besser funktionieren. In unseren Studien zeigen wir, dass Gen-Transfer in die Gesichts-Knorpel tritt effizient in einem frühen Stadium (28), wenn das Gesicht Primordium noch von Weichgewebe umfasst vor Knorpel Differenzierung. Xenopus ist ein sehr zugänglicher Wirbeltier-System für die Analyse von kraniofazialen Entwicklung. Kraniofazialen Strukturen sind gut sichtbar in Xenopus als in jedem anderen Wirbeltier-Modell, vor allem weil Xenopus Embryonen extern befruchtet werden mit Hilfe von Analysen der frühesten Stadien, und die Erleichterung leben Bildgebung bei einzelnen Zelle Auflösung, sowie die Wiederverwendung der Mütter 14. Unter Wirbeltieren Modelle entwickeln extern, ist Xenopus mehr nützlich für die kraniofaziale Analyse als Zebrafisch, wie Xenopus Larven größer und leichter zu dissect und die Entwicklung von Gesichts-Region besser zugänglich zu Bildgebung als die entsprechende Region in Fisch. Darüber hinaus ist Xenopus evolutionär näher an den Menschen als Zebrafisch (~ 100.000.000 Jahre näher) 15. Schließlich in diesen Phasen sind Xenopus Kaulquappen transparent, und die gleichzeitige Expression von fluoreszierenden Proteinen oder Molekülen wird eine einfache Visualisierung der Entwicklung von Knorpeln zu ermöglichen. Wir gehen davon aus, dass dieser Ansatz wird es uns ermöglichen, schnell und effizient Testkandidat Moleküle in einem in vivo Modellsystem.

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

Electroporation of Craniofacial Mesenchyme

Craniofacial Knorpel entwickeln in engem Kontakt mit anderen Geweben und sind schwer zu lebenden Tieren zu manipulieren. Wir sind mit Elektroporation, um molekulare Werkzeuge während des Wachstums des kraniofazialen Skeletts liefern unter Umgehung der frühen Embryonalentwicklung Effekte. Dieser Ansatz erlaubt es uns, effizient Testkandidat Moleküle<em&gt; In vivo.</em

Elektroporation von Craniofacial Mesenchym

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 Aufrufe.  King's College London. Craniofacial Knorpel entwickeln in engem Kontakt mit anderen Geweben und sind schwer zu lebenden Tieren zu manipulieren. Wir sind mit Elektroporation, um molekulare Werkzeuge während des Wachstums des kraniofazialen Skeletts liefern unter Umgehung der frühen Embryonalentwicklung Effekte. Dieser Ansatz erlaubt es uns, effizient Testkandidat Moleküle In vivo.

Serie: 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.

Elektroporation von Mykobakterien

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

Forschung

Biologie

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Antikörper Profiling von Luciferase Immunpräzipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

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.

Die Beurteilung Signaling Eigenschaften von ektodermale Epithel Während Craniofacial Entwicklung

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

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.

Eine Explantation Assay zur Beurteilung Cellular Verhalten des Cranial Mesenchym

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.

Genomweiten Gen-Deletionen in<em&gt; Streptococcus sanguinis</em&gt; Von 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 ...

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.

Trennung von embryonalen Maus-Facial Ektoderm und Mesenchym

 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.

Visualisierung von Craniofacial Entwicklung in den sox10: kaede transgener Zebrafische Zeile mit Zeitraffer-konfokale Mikroskopie

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.

Analyse Craniofacial Morphogenese in Zebrafisch Mit 4D konfokale Mikroskopie

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.

Alternative Kulturen für menschlichen pluripotenten Stammzellen Produktion, Wartung und Genetic Analysis

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.

Die Beurteilung artspezifische Um Beiträge Craniofacial Entwicklung mit Wachtel-Ente Chimären

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