Siamo grati a Nancy Papalopulu e Boyan Bonev per l&#39;assistenza con elettroporazione Xenopus. Ringraziamo anche Marc Dionne per la lettura critica, Jeremy Green e John Wallingford per utili discussioni ed i membri del laboratorio Liu per il loro sostegno. Questo lavoro è stato finanziato da sovvenzioni da parte del BBSRC (BB/E013872/1) e il Wellcome Trust (081880/Z/06/Z) per KJL.

Parte 1A. Attrezzatura Microscopio: verticale portata stereo-dissezione con obiettivo a basso <ul><li> Tensione / generatore di impulsi: BTX ECM 830 Piazza del sistema Elettroporazione Onda </li><li> Pipette puller: P-87 Micropipetta Puller (Instrument Company Sutter, CA) </li><li> Manipolatore: grossolani, o combinata grossa e fine a seconda preparazione. </li><li> Micropipetta titolare: Strumenti Scienza fine </li><li> Elettrodo: fatto in casa </li><li> Elettroporazione camera: fatto in casa </li></ul> L a forma di elettrodi: <ol><li> Tagliare 8 cm di elevata purezza filo di tungsteno 0,4 mm (Goodfellow) e apporre a metà di una siringa da 1 ml con stucco (usiamo Blu-Tack). Lasciare 4 fili di tungsteno centimetri esposti dalla punta della siringa e la punta piegare in forma di L, 1 cm dalla fine (Fig. 1A). </li><li> Trim punta in modo che alla fine le misure 0,5 millimetri di lunghezza. Tla sua punta è il capolinea degli elettrodi. </li><li> Esegui eccesso parallelo filo di tungsteno di siringa e utilizzarlo per collegare il generatore di impulsi elettrodi. </li><li> Ripetere il processo di fare una coppia di elettrodi. </li><li> Collegare gli elettrodi al generatore di impulsi quadrati onda via cavo DC. </li></ol> Camera di elettroporazione <ol><li> Bottom line di 90 mm con piatto ~ 5 mm non tossica plastilina. </li><li> Riempire piatto con i media elettroporazione. </li><li> Utilizzando n. 5 orologeria forcipe ritagliarsi una forma di T e (Fig. 2). Il pozzo lungo deve misurare ~ 2 mm x 2 mm x 10 mm e la breve ~ 2 mm x 2 mm x 5 mm. Il piatto elettroporazione può essere lavato e riutilizzato. </li></ol> Parte 1B. Reagenti DNA o macromolecole paga <ul><li> Micropipette: 1 mm di larghezza 4 &quot;lungo capillari di vetro borosilicato (WPI, TW100-F) </li><li> Cultura dei media: Normale Amphibian Media (NAM) </li><li> &gt; 10 X magazzino: 1100 mM NaCl, 20mM KCL, 10 mM Ca (NO 3) 2 • 4H 2 O, 1 mM EDTA. <ul><li> Autoclave e conservare a 4 ° C. </li><li> 1 X NAM: diluire da 10x magazzino, buffer con 0,1 mM NaHCO 3 e 0,2 mM Na 3 PO 4. </li></ul></li><li> Xenopus laevis girini, tappa 28 </li></ul> DNA preparazione: <ol><li> Preparare plasmidi di espressione utilizzando protocolli standard. </li><li> Risospendere il DNA ad una concentrazione finale di 1 mg / mL nel priva di nucleasi H 2 0. </li></ol> * Abbiamo avuto successo con i vettori che contiene un promotore forte CMV, come pCS2 + [16]. Per l&#39;analisi lignaggio, di solito includono DNA che codifica una proteina fluorescente verde (GFP + pCS2) ad una concentrazione finale di 0,1 mg / mL. Concentrazioni di DNA [tra 0,1-3 mg / mL sono stati testati. Abbiamo trovato che le concentrazioni di sotto di 0.8 mg / mL cellule inefficiente etichettati, mentre le concentrazioni di DNA superiore a 2 mg / m &amp;u, l non ha migliorato l&#39;efficienza elettroporazione]. Morfolino preparazione oligonucleotidi: (Nota:. MOS devono essere fluoresceinated (3&#39;-carboxyfluorescein modificati) o altrimenti a pagamento) <ol><li> Risospendere oligonucleotidi morfolino (MOS) (Genetools, <a href="http://www.genetools.com" target="_blank">www.genetools.com</a> ) ad una concentrazione di 2 mm priva di nucleasi H 2 0. </li><li> Aliquota di calore di soluzione a 65 ° C per 5 minuti. </li><li> Diluire a concentrazione finale di 0,5 mM in acqua priva di nucleasi. </li></ol> * 0.1-1mm MO soluzioni sono state testate. 0.5 mM soluzioni MO erano sufficienti per l&#39;elettroporazione di molte cellule mesenchimali. Micropipette <ol><li> Preparare micropipette da capillari di vetro borosilicato (1 mm di larghezza, 4 &quot;a lungo, WPI no. TW100-F). Estrattore Utilizzare l&#39;ago per preparare micropipette con un cono 8-12 mm di lunghezza e punta fine. </li><li> Crush punta ~ 2 mmdalla punta con pinze, creando una rottura frastagliato. </li></ol> Media <ol><li> Mezzi di incubazione: Preparare fresco 3 / 4 Normale Anfibio Media (NAM) da 1x magazzino. Aggiungi 0,025 mg / ml di gentamicina. </li><li> Mezzi di elettroporazione: come sopra, con il 0,1% Benzocaina (Sigma, 06950). </li></ol> 2. Elettroporazione Micropipetta di setup <ol><li> Riempire con micropipetta ~ 1 soluzione iniettabile microlitri. </li><li> Sicuro micropipetta in micromanipolatore e allegare alla microinjector (Picospritzer II). </li><li> Micropipetta angolo a 50 ° dal piano del tavolo. </li><li> Impostare la pressione di iniezione a 20 PSI. </li><li> Calibrare micropipetta per iniettare 30 nl per impulso. </li></ol> Girino preparazione <ol><li> Anestetizzare tappa 28 larve di Xenopus incubando elettroporazione in media per 5 minuti. </li><li> Trasferimento girino anestetizzato nella camera di elettroporazione riempito con i media elettroporazione. Posizione embrioneall&#39;interno del lungo e in modo che la testa resti in incrocio a T con lato dorsale verso il basso e lato ventrale esposti. La testa deve essere leggermente elevata rispetto alla coda. </li><li> Utilizzando pinze, dolcemente sicuro girino bene con circostante plastilina. (Nota:.. Se il girino non è protetto, essa può contrarsi e di contatto degli elettrodi durante elettroporazione In questo caso scartare il girino come tessuti facciali sarà gravemente danneggiato) </li></ol> Elettroporazione <ol><li> Inserire il puntale micropipetta immediatamente posteriore alla ghiandola cemento e in mesenchima del viso. </li><li> Iniettare 30 soluzione nl in mesenchima. </li><li> Ritrarre micropipetta. </li><li> Allineare rapidamente punte degli elettrodi in parallelo alla testa di un embrione (Fig. 3). </li><li> Applicare 8 50 ms, impulsi 20mV quadrati. </li><li> Ritrarre elettrodi. </li><li> Utilizzando pinze attentamente rilascio da girino bene e il trasferimento a 3 / 4 NAM, 0,025 mg / ml di gentamicina. </li><li> Girini possono essere incubate in 3 / 4 NAM, 0,025 mg / ml overnight, o più. </li><li> Embrioni schermo per l&#39;elettroporazione efficiente mediante microscopia a fluorescenza, dopo 24 ore. </li></ol> 3. Rappresentante dei risultati: L&#39;uso di molecole fluorescenti permette lo screening degli embrioni elettroporate facile. La Figura 4 mostra una partita tipica di MO elettroporate girini ~ 12, 48 e 96 ore dopo l&#39;elettroporazione, incubate a 14,5 ° C. Usando la microscopia a fluorescenza, MOS possono essere visualizzati immediatamente dopo l&#39;elettroporazione e persistono per diversi giorni dopo l&#39;elettroporazione. Nella nostra esperienza, la fluorescenza è debolmente evidente allo stadio 46 (~ 5 giorni dopo). Nel cartilagini, fluorescenza diminuisce drasticamente dopo l&#39;inizio della differenziazione (~ ° 42), tuttavia, MO fluorescenza persiste più forte in altri tipi di cellule come l&#39;endoderma faringeo. Microscopia a fluorescenza mostra che oligonucleotidi sono incorporati in vari tessuti tra cui la cartilagine cranio-facciale. Oligonucleotide fluorescenza cuno spesso essere visualizzati in tessuto su entrambi i lati della testa. Ciò è probabilmente dovuto alla rapida diffusione della soluzione iniettabile di tutto il mesenchima craniofacciale sciolto prima di elettroporazione. <img alt="Figura 1" src="/files/ftp_upload/3381/3381fig1.jpg" /> Figura 1 elettrodi fatti in casa. L a forma di filo di tungsteno è attaccato ad una siringa da 1 ml con atossici argilla o mastice. (A) Il capolinea elettrodo misure di 5 mm. (B) Allegare una coppia di elettrodi, in modo che i termini corrono parallele. Gli elettrodi sono collegati al generatore di impulsi da cavi DC. <img alt="Figura 2" src="/files/ftp_upload/3381/3381fig2.jpg" /> Figura 2 camera di elettroporazione. (A) 90 millimetri piatto rivestito con la plastilina è pieno di media e una T-camera a forma scolpita con una pinza n. 5 orologeria. (B) Il lato lungo misura 2 mm x 2 mm X10 millimetromentre le misure a breve 2 mm x 2 mm x 5 mm. La testa dell&#39;embrione riposa nella incrocio a T, lato ventrale in su. <img alt="Figura 3" src="/files/ftp_upload/3381/3381fig3.jpg" /> Figura 3 procedura di elettroporazione schematica illustrazione. St. 28 girino viene posto in camera di elettroporazione, lato ventrale in su. Micropipetta viene inserito in viso ghiandola cemento mesenchima sottostante. Iniettare. Micropipetta viene rimossa e a forma di L elettrodi sono allineati parallelamente ai lati della testa. Applicare otto 50 ms, 20 mV impulsi quadrati. Ritrarre elettrodi. Girini crescono a fasi desiderato. Visualizzate MOS o espressione GFP usando la microscopia a fluorescenza. <img alt="Figura 4" src="/files/ftp_upload/3381/3381fig4.jpg" /> Figura 4 girini Rappresentante 12 (A), 48 (B) e 96 (C) ore dopo l&#39;elettroporazione (tappe 30, 34 e 44 rispettivamente). (A &quot;B&quot;) fluorescente MO possono essere visualizzati all&#39;interno di mesenchima craniofacciale al cervoES 30 e 34. Fluorescenza possono essere rilevati in fase di cartilagini a 44 (punta di freccia, C &#39;C &quot;). L&#39;intestino è altamente autofluorescenti.

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Gli autori non hanno conflitti di interesse.

In questo video, abbiamo dimostrato la fattibilità di elettroporazione-mediata consegna del gene nel mesenchima facciale di girini Xenopus. Usando questo approccio, siamo in grado di bypassare primi effetti sullo sviluppo di manipolare la funzione del gene che ci permette di indirizzare specifici tessuti nei punti più tardi. I nostri studi mostrano che la popolazione eterogenea di cellule mesenchimali cranio-facciale può essere influenzata, che ci permette di esaminare stirpe di cellule elettroporate così come cellula autonoma requisiti per le proteine ​​di interesse. In combinazione con immagini dal vivo, possiamo usare questo approccio per studiare la funzione dei geni, nel tempo, durante lo sviluppo cranio-facciale. Questo nuovo metodo evidenzia la trattabilità di Xenopus per lo studio della organogenesi. Possiamo anticipare che questo metodo può essere ampiamente adattato per lo studio della morfogenesi e la differenziazione dei tessuti anche altri.

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;elettroporazione è un metodo efficace di fornire DNA e altre macromolecole carica nei tessuti in punti temporali precisi e in luoghi precisi. Per esempio, elettroporazione è stato utilizzato con successo per studiare lo sviluppo neurale e della retina in Xenopus, pollo e topo 1-10. Tuttavia, è importante notare che in tutti questi studi, gli investigatori non sono stati targeting tessuti molli. Perché siamo interessati allo sviluppo craniofacciale, abbiamo adattato un metodo per indirizzare mesenchima del viso. Quando abbiamo cercato la letteratura, abbiamo trovato, con nostra sorpresa, rapporti molto pochi di trasferimento genico di successo nel tessuto cartilagineo. La maggior parte di questi studi sono stati studi sulla terapia genica, come la consegna di siRNA o proteine ​​in linee cellulari condrogenico, o, modelli animali di artrite 11-13. In altri sistemi, come il pollo o il mouse, elettroporazione di mesenchima viso è stato impegnativo (COMUNICAZI personaleioni, Dipartimento dello Sviluppo cranio-facciali, KCL). Abbiamo ipotizzato che nei tessuti elettroporazione procartilaginous e cartilagineo in Xenopus potrebbe funzionare meglio. Nei nostri studi, abbiamo dimostrato che il trasferimento genico nelle cartilagini del viso si verifica in modo efficiente nelle fasi iniziali (28), quando il primordio facciale è ancora costituito da tessuti molli prima della differenziazione della cartilagine. Xenopus è un sistema molto accessibile vertebrati per l&#39;analisi dello sviluppo cranio-facciale. Strutture cranio-facciali sono più facilmente visibili in Xenopus che in qualsiasi altro modello vertebrato, in primo luogo perché gli embrioni sono fecondati esternamente Xenopus, consentendo analisi delle prime fasi, e di facilitare l&#39;imaging dal vivo a risoluzione singola cellula, nonché il riutilizzo delle madri 14. Tra i modelli vertebrati sviluppo esternamente, Xenopus è più utile per l&#39;analisi del cranio di pesce zebra, come larve di Xenopus sono più grandi e più facile dissect, e la regione lo sviluppo del viso è più accessibile per l&#39;imaging rispetto alla regione equivalente nel pesce. Inoltre, Xenopus è evolutivamente più vicina agli esseri umani di zebrafish (~ 100 milioni anni più vicini) 15. Infine, in queste fasi, girini Xenopus sono trasparenti, e l&#39;espressione contemporanea di proteine ​​fluorescenti o molecole permette una facile visualizzazione delle cartilagini di sviluppo. Possiamo anticipare che questo approccio ci permetterà di testare rapidamente ed efficacemente le molecole candidato in un sistema modello in vivo.

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

Electroporation of Craniofacial Mesenchyme

Cartilagini craniofacciali sviluppare a stretto contatto con altri tessuti e sono difficili da manipolare di animali vivi. Stiamo usando elettroporazione per fornire strumenti molecolari durante la crescita dello scheletro cranio-facciale, mentre bypassando primi effetti embrionale. Questo approccio ci permetterà di testare in modo efficiente molecole candidate<em&gt; In vivo.</em

Elettroporazione di Mesenchima craniofacciale

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 Views.  King's College London. Cartilagini craniofacciali sviluppare a stretto contatto con altri tessuti e sono difficili da manipolare di animali vivi. Stiamo usando elettroporazione per fornire strumenti molecolari durante la crescita dello scheletro cranio-facciale, mentre bypassando primi effetti embrionale. Questo approccio ci permetterà di testare in modo efficiente molecole candidate In vivo.

Video: 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.

Elettroporazione di micobatteri

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

Ricerca

Biologia

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.

Profiling anticorpi Systems Immunoprecipitazione luciferasi (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.

La valutazione delle proprietà di segnalazione di ectodermica epiteli durante lo sviluppo craniofacciale

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.

Un test per la valutazione dei comportamenti Espianto cellulare del Mesenchima cranio

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.

Genome-wide gene delezioni in<em&gt; Streptococcus sanguinis</em&gt; Da Throughput PCR ad alta

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.

Separazione dei Ectoderma embrionali di topo viso e Mesenchima

 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.

Visualizzazione di sviluppo craniofacciale nei SOX10: Kaede transgenici Zebrafish line utilizzando Time-lapse Microscopia 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.

Analizzando craniofacciale morfogenesi in Zebrafish Uso 4D Microscopia 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.

Le colture per pluripotenti umane alternative staminali produzione di celle, manutenzione e analisi genetica

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.

Valutare Contributi specie-specifiche per lo sviluppo craniofacciale Uso Chimere Quaglia-anatra

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