Questo lavoro è stato sostenuto da un finanziamento della Fondazione per la ricerca tedesco (DFG, LU1455/1-1).

1. Cultura Ex ovo <ol><li> Freschi uova fecondate sono stabiliti sul loro lato lungo in un forzata progetto incubatore (BSS160, Ehret, Germania) a 37,5 ° C con 60% di umidità per l&#39;incubazione. Uova conservati a 12 ° C per più di una settimana non dovrebbe essere usato. </li><li> Dopo incubazione per 2,5 giorni (circa fase HH17), estrarre le uova ed etichettare la parte superiore con una matita per indicare la direzione per il cracking. </li><li> Prima di cracking, versare circa 20 ml di acqua distillata sterile in un grande piatto pulito Petri (diametro di 145 mm; Greiner Bio-One GmbH, Germania). </li><li> Posizionare un altro piccolo piastra di Petri sterile (diametro di 94 mm; Greiner Bio-One GmbH) nel grande piatto Petri. </li><li> Rompere le uova sul fondo contro un bordo di metallo affilato, aprire con cautela le uova e trasferire l&#39;intero contenuto di ciascun uovo nella piccola scatola di Petri. </li><li> Coprire il piatto grande Petri con un coperchio e metterlo in un altro incubatore (Binder GmbH, Tuttlinge, Germania)per la cultura ulteriormente a 37,5 ° C con circa il 60% di umidità. Dopo incubazione per il numero desiderato di giorni, gli embrioni possono essere utilizzati per elettroporazione. </li></ol> 2. Preparazione per l&#39;ex ovo Elettroporazione <ol><li> Pull in vetro capillari con un estrattore microelettrodo (PUL-100 Micropipetta Puller, strumenti di precisione del Mondo, Berlino, Germania) utilizzando tubi di vetro di 1,0 mm di diametro (TW100F-4; Strumenti di precisione del Mondo) e rompere la punta dei capillari di vetro in un apposito diametro. </li><li> Impostare i parametri appropriati (ad esempio, tensioni diverse per i diversi tessuti e dimensioni di embrioni) per l&#39;elettroporazione e collegare gli elettrodi a un elettroporatore (CUY21-Edit, Nepa Gene, Chiba, Giappone) in base ai tessuti bersaglio e le dimensioni di embrioni. </li><li> Preparare una soluzione plasmide contenente plasmidi a vettore pCAGGS codificano un gene bersaglio, ad es cadherin7 (Cad7; pCAGGS-Cad7 con una concentrazione di 2,0 pg / pl) e un gene marcatore,ad esempio, proteina verde fluorescente (GFP; pCAGGS-GFP con una concentrazione di 0,25 mg / pl). Poi aggiungere verde veloce con una concentrazione finale di 0,1% (Sigma) per marcare la soluzione plasmide con il colore verde. </li><li> Caricare il capillare di vetro con la soluzione plasmide usando una pipetta a bocca. </li></ol> 3. Il trasferimento genico in Tectum pollo ottica da ex ovo Elettroporazione <ol><li> Dopo coltura ex ovo, ad esempio, di giorno incubazione (E) 6, gli embrioni di pollo sono utilizzati per esempio elettroporazione ovo. </li><li> Attenzione allo strappo le membrane amnios vitellina e il tetto ottico con una pinza sottili e aggiungere 3 gocce di soluzione sterile con sodio cloruro 0,9% sulla superficie del Tectum. </li><li> Iniettare la soluzione plasmide nella cavità del tectum dal capillare di vetro usando la pipetta bocca. </li><li> Posizionare gli elettrodi con un catodo ago di tungsteno e un anodo di piastra rettangolo (Cuy 661-3x7, Nepa Gene) accanto al Tectum eapplicare immediatamente impulsi elettrici (sei impulsi, 25 V, 60 ms lunghezza di impulso, a intervalli di 100 ms in ogni caso) prodotte dalla elettroporatore al Tectum. </li><li> Coprire il piatto Petri grande con il coperchio e restituirla nell&#39;incubatore. Dopo incubazione per giorno appropriate (ad esempio, 2 o 3 giorni dopo l&#39;elettroporazione), il tectum sono raccolti e fissati per immunocolorazione (figura 1) e il rilevamento biologico. </li></ol> 4. Risultati rappresentativi Sovraespressione successo delle proteine ​​esogene di Cad7 e GFP da ex elettroporazione ovo è illustrato nella figura 1 come esempio. Quando Cad7 insieme plasmide con GFP plasmide è stato elettroporata in tectum a E6, due o tre giorni dopo l&#39;elettroporazione gli embrioni sono stati raccolti e fissati. Al E8, la proteina GFP è fortemente espresso nel tetto in intere immagini montaggio (Figura 1A-C). Inoltre, nelle sezioni del tectum a E9,Proteina GFP (verde in figura 1D) e Cad7 proteina (rosso in Figura 1E) sono coespressa (giallo in figura 1F), suggerendo che ex elettroporazione ovo è un metodo efficace per il trasferimento genico in gli embrioni di pollo anziani in vivo. <img src="/files/ftp_upload/4078/4078fig1.jpg" alt="Figura 1"/> Figura 1. Quando il plasmidi codifica GFP e Cad7 sono cotransfected nel tectum pollo a E6, due o tre giorni dopo l&#39;elettroporazione ex ovo, proteina GFP (verde) è fortemente espresso come mostrato in immagini wholemount (AC) a E8. Nelle sezioni del tectum a E9, proteina GFP (green in D) e Cad7 proteine ​​(rosso in E) sono coespressi (giallo in F). BF, immagine campo chiaro. Scale bar: 200 micron in A per AC; 100 micron di D per DF.

<ol>
	<li>Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K., Yasuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+gene+expression+in+chick+embryos+by+microelectroporation.">Efficient targeting of gene expression in chick embryos by microelectroporation.</a> Dev. Growth Differ. 41, 335-344 (1999).</li><li>Luo, J., Ju, M. J., Lin, J., Yan, X., Markus, A., Mix, E., Rolfs, A., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-20+expression+by+motor+neurons+is+regulated+by+Sonic+hedgehog+during+spinal+cord+development.">Cadherin-20 expression by motor neurons is regulated by Sonic hedgehog during spinal cord development.</a> NeuroReport. 20, 365-370 (2009).</li><li>Luo, J., Ju, M. J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regionalized+cadherin-7+expression+by+radial+glia+is+regulated+by+Shh+and+Pax7+during+chicken+spinal+cord+development.">Regionalized cadherin-7 expression by radial glia is regulated by Shh and Pax7 during chicken spinal cord development.</a> Neuroscience. 142, 1133-1143 (2006).</li><li>Nakamura, H., Katahira, T., Sato, T., Watanabe, Y., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain-+and+loss-of-function+in+chick+embryos+by+electroporation.">Gain- and loss-of-function in chick embryos by electroporation.</a> Mech. Dev. 121, 1137-1143 (2004).</li><li>Dai, F., Yusuf, F., Farjah, G. H., Brand-Saberi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-induced+targeted+silencing+of+developmental+control+genes+during+chicken+embryogenesis.">RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis.</a> Dev. Biol. 258, 80-90 (2005).</li><li>Rao, M., Baraban, J. H., Rajaii, F., Sockanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+comparative+study+of+RNAi+methodologies+by+in+ovo+electroporation+in+the+chick+embryo.">In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo.</a> Dev. Dyn. 231, 592-600 (2004).</li><li>Kos, R., Tucker, R. P., Hall, R., Duong, T. D., Erickson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+introducing+morpholinos+into+the+chicken+embryo.">Methods for introducing morpholinos into the chicken embryo.</a> Dev. Dyn. 226, 470-477 (2003).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> J. Morphol. 88, 49-92 (1951).</li><li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev. Biol. 10, 391-394 (1974).</li><li>Luo, J., Treubert-Zimmermann, U., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherins+guide+migrating+Purkinje+cells+to+specific+parasagittal+domains+during+cerebellar+development.">Cadherins guide migrating Purkinje cells to specific parasagittal domains during cerebellar development.</a> Mol. Cell. Neurosci. 25, 138-152 (2004).</li><li>Luo, J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+ovo+electroporation+for+gene+transfer+into+older+chicken+embryos.">Ex ovo electroporation for gene transfer into older chicken embryos.</a> Dev. Dyn. 233, 1470-1477 (2005).</li><li>Treubert-Zimmermann, U., Heyers, D., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+axons+to+specific+fiber+tracts+in+vivo+by+altering+cadherin+expression.">Targeting axons to specific fiber tracts in vivo by altering cadherin expression.</a> J. Neurosci. 22, 7617-7626 (2002).</li></ol>

Non ci sono conflitti di interesse dichiarati.

Questo protocollo fornisce una guida per il trasferimento del gene in embrioni di pollo grandi (ad esempio, nel tetto ottico a E6) di elettroporazione ex ovo. Questo metodo estende l&#39;utilizzo di elettroporazione di embrioni di pollo anziani e può essere facilmente applicato a molti laboratori per lo studio della funzione genica in vivo in fasi successive. Gli embrioni da E4 ad almeno E7 può essere successo elettroporate con questo metodo e gli embrioni possono essere elettroporate sopravvivenza f...

<table border="1"><tbody><tr><td> Nome </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Elettroporatore </td><td> Nepa Gene, Giappone </td><td> CUY21-Edit </td></tr><tr><td> Elettrodi </td><td> Nepa Gene, Giappone </td><td> CUY661-3x7 </td></tr><tr><td> Egg incubatore </td><td> Ehret, Germania </td><td> BSS160 </td></tr><tr><td> Embryo incubatore </td><td> BINDER GmbH, Germany </td><td></td></tr><tr><td> Microscopio a fluorescenza </td><td> Keyence, Deutschland GmbH </td><td> BZ-8000 </td></tr><tr><td> Petri </td><td> Greiner Bio-One GmbH, Germania </td><td></td></tr><tr><td> Microelettrodo puller </td><td> Mondiale strumenti di precisione, Germania </td><td> PUL-100 </td></tr></tbody></table>

gene transfer into older chicken embryos by ex ovo electroporation

L&#39;embrione di pollo fornisce un ottimo sistema modello per studiare la funzione del gene e la regolazione durante lo sviluppo embrionale. In elettroporazione ovo è un metodo efficace per geni esogeni over-espressa o down-regolare i geni endogeni in vivo in embrioni di pollo 1. Di strutture diverse come i geni codificanti del DNA plasmidi 2-4, piccoli RNA interferenti (siRNA) 5 plasmidi, piccoli RNA sintetico oligos 6 e 7 morpholino oligonucleotidi antisenso può essere facilmente trasfettati in embrioni di pollo mediante elettroporazione. Tuttavia, l&#39;applicazione di elettroporazione in ovo è limitata a embrioni nelle fasi iniziali di incubazione (meno di stage HH20 - in base ad Amburgo e Hamilton) 8 e ci sono alcuni svantaggi per la sua applicazione in embrioni in fasi successive (più vecchio di stage HH22 - circa 3,5 giorni di sviluppo). Ad esempio, la membrana vitellina in fasi successive è solitamentealleato attaccato alla membrana deve e aprendo una finestra nel guscio provoca la rottura dei vasi, con conseguente morte degli embrioni; vecchi embrioni sono coperti da navi vitellina e allantoico, dove è difficile accedere e manipolare gli embrioni; vecchi embrioni spostare vigorosamente ed è difficile controllare l&#39;orientamento attraverso una finestra relativamente piccola nel guscio. In questo protocollo si dimostra un metodo di elettroporazione ex ovo per il trasferimento del gene in embrioni di pollo nelle fasi tardive (di età superiore a stage HH22). Per elettroporazione ex ovo, gli embrioni vengono coltivati ​​in piastre di Petri 9 e il vitellino e vasi allantoide sono ampiamente diffuse. In queste condizioni, gli embrioni di pollo anziani sono facilmente accessibili e manipolati. Pertanto, questo metodo supera gli svantaggi della elettroporazione in ovo applicata ai vecchi embrioni di pollo. Utilizzando questo metodo, plasmidi possono essere facilmente trasfettato in parti differentidegli embrioni di pollo anziani 10-12.

Watch this Scientific Journal Video about Gene Transfer into Older Chicken Embryos by ex ovo Electroporation at JoVE.com

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

Un metodo di trasferimento genico in embrioni di pollo in fasi successive di incubazione (più di Hamburger e Hamilton stadio (HH) 22) è descritto. Questo metodo supera inconvenienti<em&gt; In ovo</emElettroporazione&gt; applicata a embrioni di pollo anziani ed è una tecnica utile per studiare la funzione regolazione dei geni e alle vecchie stadi di sviluppo.

Il trasferimento genico in embrioni di pollo da anziani<em&gt; Ex ovo</em&gt; Elettroporazione

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

gene-transfer-into-older-chicken-embryos-by-ex-ovo-electroporation

Research

JoVE Journal

Biology

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

14.5K Views.  School of Medicine University of Rostock. Un metodo di trasferimento genico in embrioni di pollo in fasi successive di incubazione (più di Hamburger e Hamilton stadio (HH) 22) è descritto. Questo metodo supera inconvenienti In ovo applicata a embrioni di pollo anziani ed è una tecnica utile per studiare la funzione regolazione dei geni e alle vecchie stadi di sviluppo.

Video: Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

Trasferimento nucleare in oociti mouse

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

Ricerca

Biologia

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

In Utero iniezione intraventricolare e Elettroporazione di embrioni di ratto E16

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In Utero iniezione intraventricolare e Elettroporazione di E15 embrioni di topo

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

Posizionamento Growth Factor-Coated Perline su embrioni di pollo Stadio

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

In Electroporations Ovo della Fase HH 10 embrioni di pollo

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Microiniezione di embrioni di zebrafish per analizzare la funzione del gene

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

In elettroporazione in vivo ed ex utero per l&#39;espressione genica in cellule gangliari della retina del mouse

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Pulcino Ex ovo Cultura e Ex ovo CAM saggio: Come funziona veramente

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Il trasferimento genico in embrioni di pollo da anziani

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