Name: technique to target microinjection to the developing xenopus kidney
Uploaded: 2016-05-03T11:00:00
Description: Watch this Scientific Journal Video about Technique to Target Microinjection to the Developing Xenopus Kidney at JoVE.com

Diese Arbeit wurde von einem National Institutes of Health NIDDK Zuschuss (K01DK092320) und Anschubfinanzierung von der Abteilung für Pädiatrie an der University of Texas Medical School McGovern unterstützt.

(: HSC-AWC-13-135-Protokoll #) Das folgende Protokoll wurde von der University of Texas Health Science Center in Houston Center for Laboratory Animal Medicine Animal Welfare Committee, das dient als Institutional Care und Verwenden Committee genehmigt. 1. Identifizierung und Auswahl von Blastomeren für Injektionszwecke Nieren-bezogenen <ol><li> Vor der Embryonen zu erzeugen, verwenden Sie die normale Tabelle der Xenopus - Entwicklung 21 , um die Ausrichtung der ersten Zellteilungen im Embr...

<ol>
	<li>Vize, P. D., Seufert, D. W., Carroll, T. J., Wallingford, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+systems+for+the+study+of+kidney+development:+Use+of+the+pronephros+in+the+analysis+of+organ+induction+and+patterning.">Model systems for the study of kidney development: Use of the pronephros in the analysis of organ induction and patterning.</a> Dev. Biol. 188 (2), 189-204 (1997).</li><li>Brandli, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+molecular+anatomy+of+the+Xenopus+pronephric+kidney.">Towards a molecular anatomy of the Xenopus pronephric kidney.</a> Int. J. Dev. Biol. 43 (5), 381-395 (1999).</li><li>Hensey, C., Dolan, V., Brady, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xenopus+pronephros+as+a+model+system+for+the+study+of+kidney+development+and+pathophysiology.">The Xenopus pronephros as a model system for the study of kidney development and pathophysiology.</a> Nephrol. Dial. Transplant. 17, 73-74 (2002).</li><li>Carroll, T., Vize, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergism+between+Pax-8+and+lim-1+in+embryonic+kidney+development.">Synergism between Pax-8 and lim-1 in embryonic kidney development.</a> Dev. Biol. 214 (1), 46-59 (1999).</li><li>Zhou, X., Vize, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximo-distal+specialization+of+epithelial+transport+processes+within+the+Xenopus+pronephric+tubules.">Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric tubules.</a> Dev. Biol. 271 (2), 322-338 (2004).</li><li>Raciti, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+pronephric+kidney+revealed+by+large-scale+gene+expression+mapping.">Organization of the pronephric kidney revealed by large-scale gene expression mapping.</a> Genome Biol. 9 (5), 84 (2008).</li><li>Moody, S. A., Kline, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+fate+during+cleavage+of+frog+(Xenopus+laevis)+blastomeres.">Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres.</a> Anat. Embryol. Berl. 182 (4), 347-362 (1990).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+16-cell+stage+of+Xenopus+embryo.">Fates of the blastomeres of the 16-cell stage of Xenopus embryo.</a> Dev. Biol. 119 (2), 560-578 (1987).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+32-cell+stage+of+Xenopus+embryo.">Fates of the blastomeres of the 32-cell stage of Xenopus embryo.</a> Dev. Biol. 122 (2), 300-319 (1987).</li><li>Bauer, D. V., Huang, S., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cleavage+stage+origin+of+Spemann's+Organizer:+Analysis+of+the+movements+of+blastomere+clones+before+and+during+gastrulation+in+Xenopus.">The cleavage stage origin of Spemann's Organizer: Analysis of the movements of blastomere clones before and during gastrulation in Xenopus.</a> Dev. 120 (5), 1179-1189 (1994).</li><li>Karpinka, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenbase,+the+Xenopus+model+organism+database;+new+virtualized+system,+data+types+and+genomes.">Xenbase, the Xenopus model organism database; new virtualized system, data types and genomes.</a> Nucleic Acids Res. 43, 756-763 (2015).</li><li>Davidson, L. A., Marsden, M., Keller, R., Desimone, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+alpha5beta1+and+fibronectin+regulate+polarized+cell+protrusions+required+for+Xenopus+convergence+and+extension.">Integrin alpha5beta1 and fibronectin regulate polarized cell protrusions required for Xenopus convergence and extension.</a> Curr. Biol. 16 (9), 833-844 (2006).</li><li>Wallingford, J. B., Carroll, T. J., Vize, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precocious+expression+of+the+Wilms'+tumor+gene+xWT1+inhibits+embryonic+kidney+development+in+Xenopus+laevis.">Precocious expression of the Wilms' tumor gene xWT1 inhibits embryonic kidney development in Xenopus laevis.</a> Dev. Biol. 202, 103-112 (1998).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early Development of Xenopus laevis. A Laboratory Manual. , (2000).</li><li>Miller, R. K., Lee, M., McCrea, P. D., Kloc, M., Kubiak, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xenopus+pronephros:+A+kidney+model+making+leaps+toward+understanding+tubule+development.">The Xenopus pronephros: A kidney model making leaps toward understanding tubule development.</a> Xenopus Development. , 215-238 (2014).</li><li>Lienkamp, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+kidney+tubules+elongate+using+a+planar+cell+polarity-dependent+rosette-based+mechanism+of+convergent+extension.">Vertebrate kidney tubules elongate using a planar cell polarity-dependent rosette-based mechanism of convergent extension.</a> Nat. Genet. 44 (12), 1382-1387 (2012).</li><li>Lyons, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+Wnt/&#946;-catenin+signaling+in+pronephric+kidney+development.">Requirement of Wnt/&#946;-catenin signaling in pronephric kidney development.</a> Mech. Dev. 126 (3-4), 142-159 (2009).</li><li>Miller, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pronephric+tubulogenesis+requires+Daam1-mediated+planar+cell+polarity+signaling.">Pronephric tubulogenesis requires Daam1-mediated planar cell polarity signaling.</a> J. Am. Soc. Nephrol. 22, 1654-1667 (2011).</li><li>Vize, P. D., Jones, E. A., Pfister, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Xenopus+pronephric+system.">Development of the Xenopus pronephric system.</a> Dev. Biol. 171 (2), 531-540 (1995).</li><li>Miller, R. K., Lee, M., McCrea, P. D., Kloc, M., Kubiak, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+12:+The+Xenopus+Pronephros:+A+Kidney+Model+Making+Leaps+toward+Understanding+Tubule+Development.">Chapter 12: The Xenopus Pronephros: A Kidney Model Making Leaps toward Understanding Tubule Development.</a> Xenopus Development. , (2014).</li><li>Taira, M., Otani, H., Jamrich, M., Dawid, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+LIM+class+homeobox+gene+Xlim-1+in+pronephros+and+CNS+cell+lineages+of+Xenopus+embryos+is+affected+by+retinoic+acid+and+exogastrulation.">Expression of the LIM class homeobox gene Xlim-1 in pronephros and CNS cell lineages of Xenopus embryos is affected by retinoic acid and exogastrulation.</a> Development. 120, 1525-1536 (1994).</li><li>Weber, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+function+of+the+tissue-specific+transcription+factor+HNF1+alpha+(LFB1)+during+Xenopus+development.">Regulation and function of the tissue-specific transcription factor HNF1 alpha (LFB1) during Xenopus development.</a> Int. Dev. Biol. 40 (1), 297-304 (1996).</li><li>Carroll, T. J., Vize, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergism+between+Pax-8+and+lim-1+in+embryonic+kidney+development.">Synergism between Pax-8 and lim-1 in embryonic kidney development.</a> Dev. Biol. 214 (1), 46-59 (1999).</li><li>Etkin, L. D., Pearman, B., Ansah-Yiadom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+injected+DNA+templates+in+Xenopus+embryos.">Replication of injected DNA templates in Xenopus embryos.</a> Exp. Cell Res. 169 (2), 468-477 (1987).</li><li>Vize, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chloride+conductance+channel+ClC-K+is+a+specific+marker+for+the+Xenopus+pronephric+distal+tubule+and+duct.">The chloride conductance channel ClC-K is a specific marker for the Xenopus pronephric distal tubule and duct.</a> Gene Expr. Patterns. 3 (3), 347-350 (2003).</li><li>Uochi, T. S., Takahashi, S., Ninomiya, H., Fukui, A., Asashima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Na+,+K+-ATPase+alpha+subunit+requires+gastrulation+in+the+Xenopus+embryo.">The Na+, K+-ATPase alpha subunit requires gastrulation in the Xenopus embryo.</a> Dev. Growth Differ. 39 (5), 571-580 (1997).</li><li>Nieuwkoop, P. D., Faber, 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> Normal table of Xenopus laevis (Daudin): A systematical &amp; chronological survey of the development from the fertilized egg till the end of metamorphosis. , (1994).</li><li>Ubbels, G. A., Hara, K., Koster, C. H., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+functional+role+of+the+cytoskeleton+in+determination+of+the+dorsoventral+axis+in+Xenopus+laevis+eggs.">Evidence for a functional role of the cytoskeleton in determination of the dorsoventral axis in Xenopus laevis eggs.</a> J. Embryol. Exp. Morphol. 77, 15-37 (1983).</li><li>Huang, S., Johnson, K. E., Wang, H. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blastomeres+show+differential+fate+changes+in+8-cell+Xenopus+laevis+embryos+that+are+rotated+90+degrees+before+the+first+cleavage.">Blastomeres show differential fate changes in 8-cell Xenopus laevis embryos that are rotated 90 degrees before the first cleavage.</a> Dev. Growth Differ. 40 (2), 189-198 (1998).</li><li>Colman, A., Drummond, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stability+and+movement+of+mRNA+in+Xenopus+oocytes+and+embryos.">The stability and movement of mRNA in Xenopus oocytes and embryos.</a> J. Embryol. Exp. Morph. 97, 197-209 (1986).</li></ol>

Targeting die pronephros der Entwicklung von Xenopus - Embryonen stützt sich auf die Identifizierung und die richtige Blastomere zu injizieren. Die Injektion des V2 Blastomere von 8-Zellen - Embryonen zielt auf die linke pronephros 18. Dies lässt die Gegenseite rechts pronephros als interne Kontrolle. Wenn Morpholino Knockdown oder RNA-Überexpression wird verwendet, Nierenentwicklung zu verändern, können die Gegenseite rechts pronephros verwendet werden, um die Auswirkungen von Knock-Down oder ?...

Die Xenopus embryonale Niere, die pronephros, ist ein gutes Modell der Nierenentwicklung und Krankheit zu studieren. Die Embryonen entwickeln extern, sind groß, in großen Stückzahlen hergestellt werden und werden durch Mikroinjektion oder chirurgische Eingriffe leicht manipulierbar. Darüber hinaus werden die Gene regeln Nierenentwicklung in Säugetieren und Amphibien konserviert. Mammalian Nieren Fortschritte durch drei Phasen: die pronephros, Mesonephros und metanephros 1, während der embryonalen Amphibien haben ein pronephros und erwachsene Amphibien haben ein metanephros. Die grundlegende Filtereinheit dieser Nierenformen ist das Nephron, und beide Säugetiere und Amphibien benötigen , um die gleiche Signalkaskaden und induktive Ereignisse 3 Nephrogenese 2, zu unterziehen. Die Xenopus pronephros einen einzigen nephron enthält proximaler zusammengesetzt, Zwischen-, distalen und Anschluss Tubuli, und eine Glomus (analog dem Säuger Glomerulus) 1, 4-6 (1 </strOng&gt;). Die einzelne, große Nephron in den Xenopus pronephros macht es geeignet als einfaches Modell für die Untersuchung von Genen in Nierenentwicklung und Krankheitsprozessen beteiligt. Zellschicksal Karten wurden für frühen Xenopus - Embryonen hergestellt und sind frei im Internet unter Xenbase 7-11 zur Verfügung. Hier beschreiben wir ein Verfahren zur Mikroinjektion von Lineage Tracern , die Entwicklungs Xenopus pronephros zu zielen, wobei die gleiche Technik angepasst werden kann , andere Gewebe zu zielen, wie das Herz oder die Augen. Lineage Tracern sind Etiketten (einschließlich Vitalfarbstoffen, fluoreszierend markierte Dextrane, histochemisch nachweisbare Enzyme und mRNA kodiert, fluoreszierende Proteine), die in einem frühen Blastomere injiziert werden kann, so dass die Visualisierung der Nachkommen dieser Zelle während der Entwicklung. Dieses Protokoll nutzt MEM-RFP - mRNA, die Codierung Membran rot fluoreszierendes Protein 12 als Linie Tracer gezielt. Die gezielte Mikroinjektions techniken für einzelne Blastomeren in 4- und 8-Zellen-Embryonen hier beschriebene zur Injektion mit Morpholinos genutzt werden, um knock down Genexpression oder mit exogenen RNA ein Gen von Interesse überexprimiert. Durch die Injektion in die Bauch, pflanzliche Blastomere, in erster Linie die pronephros des Embryos wird gezielt werden, die kontralateralen pronephros als Entwicklungskontrolle verlassen. Co-Einspritzen eines Tracers verifiziert, dass der korrekte Blastomere injiziert wurde, und zeigt, welche in der Embryogeweben ergaben sich aus dem injizierten Blastomere, Verifizieren der pronephros Targeting. Immunostaining der pronephros ermöglicht die Visualisierung, wie gut die pronephric Tubuli gezielt wurden. Überexpression und Zuschlags Effekte können dann auf die Gegenseite des Embryos erzielt werden, die als Entwicklungskontrolle dient und verwendet werden kann , die pronephric Index 13 zu berechnen. Die Verfügbarkeit von Zellschicksal Karten ermöglicht diese gezielte Mikroinjektionstechnik verwendet werden, an Zielgewebe other als die pronephros und Co-Injektion eines fluoreszierenden Indikators erlaubt die gezielte Mikroinjektions zu jedem Gewebe vor der Analyse überprüft werden. Während der Embryomikroinjektions sollte Entwicklungstemperatur fest geregelt werden, da die Rate der Xenopus Entwicklung auf sie stark abhängig ist 14. (- 16 ° C 14) 4- und 8-Zellinjektionen, da die Entwicklungszeit verlangsamt Embryonen bei kühleren Temperaturen inkubiert werden. Bei 22 ° C, die Entwicklungszeit von Stufe 1 (1 Zelle) auf Stufe 3 (4 Zellen) beträgt etwa 2 Stunden, während bei 16 ° C Entwicklungszeit zu Stufe 3 ca. 4 Stunden. Es dauert etwa 15 Minuten von einem 4-Zellen-Embryo zu einem 8-Zellen (Stufe 4) Embryo bei 22 ° C zu gehen, aber dauert etwa 30 Minuten bei 16 ° C. In ähnlicher Weise bei 22 ° C, dauert es nur 30 Minuten für eine 8-Zell-Embryo zu einem 16-Zellen-Embryo (Stufe 5) fortzuschreiten. Diese Zeit wird bei 16 ° C auf 45 Minuten erhöht. Dasrefore, ist es nützlich, um die Entwicklungsrate der Embryonen zu verlangsamen, an der 8-Zellen-Stadium für Injektionszwecke ausreichend Zeit zu ermöglichen, bevor die Embryonen auf die Bühne 16-Zelle fortzuschreiten. Außerdem können Wachstumstemperaturen moduliert werden embryonale Entwicklung zu beschleunigen oder zu verlangsamen, bis die Niere vollständig entwickelt hat. Die Epidermis der Kaulquappe stufigen Xenopus - Embryonen ist relativ transparent, erlaubt eine einfache Abbildung der Entwicklung pronephros ohne Dissektion oder Lichtung des Gewebes 15. Aufgrund der relativen Transparenz von Xenopus - Embryonen ist die Abbildung lebender Zellen auch denkbar , 16,17. Whole-mount die pronephros zu visualisieren immunostaining ist möglich , mit etablierten Antikörper, die die proximalen, mittleren distalen und Verbindungs ​​Tubuli der Stufe 38 beschriften - 40 Embryonen , die für die Beurteilung der pronephric Entwicklung ermöglichen nach Manipulation der Genexpression gezielt in Xenopus - Embryonen 18-20.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate&#160;</td>
			<td>Fisher</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher</td>
			<td>S311-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin solution, 50 mg/mL</td>
			<td>Amresco</td>
			<td>E737-20ML</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine, 99%+</td>
			<td>Acros Organics</td>
			<td>52-90-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>Fisher</td>
			<td>BP525-100</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm x 15 mm Petri dish</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Fridge II</td>
			<td>Boekel</td>
			<td>260009</td>
			<td>Benchtop incubator for embryos.</td>
		</tr>
		<tr>
			<td>Gap43-RFP plasmid</td>
			<td></td>
			<td></td>
			<td>For making tracer RNA. Ref: Davidson et al., 2006.</td>
		</tr>
		<tr>
			<td>Rhodamine dextran, 10,000 M.W.</td>
			<td>Invitrogen</td>
			<td>D1817</td>
			<td>Tracer.</td>
		</tr>
		<tr>
			<td>Molecular biology grade, USP sterile purified water</td>
			<td>Corning</td>
			<td>46-000-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>7&#34; Drummond replacement tubes</td>
			<td>Drummond</td>
			<td>3-000-203-G/XL</td>
			<td>Microinjection needles.</td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Sutter Instruments</td>
			<td>P-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Dumont</td>
			<td>11252-30</td>
			<td>For breaking microinjection needle tip.</td>
		</tr>
		<tr>
			<td>Nanoject II</td>
			<td>Drummond</td>
			<td>3-000-204</td>
			<td>Microinjector.</td>
		</tr>
		<tr>
			<td>Mineral oil, heavy</td>
			<td>Fisher</td>
			<td>CAS 8042-47-5</td>
			<td>Oil for needles.</td>
		</tr>
		<tr>
			<td>27G monoject hypodermic needle</td>
			<td>Covidien</td>
			<td>8881200508</td>
			<td>For loading mineral oil into microinjection needle.</td>
		</tr>
		<tr>
			<td>5 mL Luer-Lok syringe</td>
			<td>BD</td>
			<td>309646</td>
			<td>For loading mineral oil into microinjection needle.</td>
		</tr>
		<tr>
			<td>60 mm x 15 mm Petri dish</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>800 micron polyester mesh</td>
			<td>Small Parts</td>
			<td>CMY-0800-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipets</td>
			<td>Fisher</td>
			<td>13-711-7M</td>
			<td>For transferring embryos. Cut off the tip so that the embryos are easily taken up by the pipette.</td>
		</tr>
		<tr>
			<td>6-well cell culture plate</td>
			<td>Nest Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Fisher</td>
			<td>BP308-500</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Acros Organics</td>
			<td>67-42-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher</td>
			<td>BP531-500</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mm x 45 mm screw thread vial</td>
			<td>Fisher</td>
			<td>03-339-25B</td>
			<td>For fixing, staining, and storing embryos.</td>
		</tr>
		<tr>
			<td>24-well cell culture plate</td>
			<td>Nest Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benzocaine</td>
			<td>Spectrum</td>
			<td>BE130</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher</td>
			<td>BP2818-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) 1X powder</td>
			<td>Fisher</td>
			<td>BP661-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumen</td>
			<td>Fisher</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>3D mini rocker, model 135</td>
			<td>Denville Scientific</td>
			<td>57281</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum, New Zealand origin</td>
			<td>Invitrogen</td>
			<td>16210064</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Fisher</td>
			<td>S227I-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal 3G8 antibody</td>
			<td>European Xenopus Resource Centre</td>
			<td></td>
			<td>Primary antibody to label proximal tubules.</td>
		</tr>
		<tr>
			<td>Monoclonal 4A6 antibody</td>
			<td>European Xenopus Resource Centre</td>
			<td></td>
			<td>Primary antibody to label distal tubule and duct.</td>
		</tr>
		<tr>
			<td>anti-RFP pAb, purified IgG/rabbit</td>
			<td>MBL</td>
			<td>PM005</td>
			<td>Primary antibody to label Gap43-RFP tracer.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG</td>
			<td>Life Technologies</td>
			<td>A11001</td>
			<td>Secondary antibody to label distal tubule and duct.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 goat anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>A21428</td>
			<td>Secondary antibody to label Gap43-RFP tracer.</td>
		</tr>
		<tr>
			<td>9 cavity spot plate</td>
			<td>Corning</td>
			<td>7220-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl benzoate</td>
			<td>Fisher</td>
			<td>105862500</td>
			<td>Optional - for clearing embryos</td>
		</tr>
		<tr>
			<td>Benzyl alcohol</td>
			<td>Fisher</td>
			<td>A396-500</td>
			<td>Optional - for clearing embryos</td>
		</tr>
	</tbody>
</table>

technique to target microinjection to the developing xenopus kidney

Die embryonale Niere von Xenopus laevis (Frosch), die pronephros, besteht aus einem einzigen Nephron, und kann als ein Modell für eine Nierenerkrankung verwendet werden. Xenopus - Embryonen sind groß, entwickeln außen und leicht durch Mikroinjektion oder chirurgische Verfahren manipuliert werden können. Darüber hinaus wurden für die frühen Xenopus - Embryonen Schicksal Karten hergestellt. Gezielte Mikroinjektion in den einzelnen Blastomeren, die Anlass schließlich zu einem Organ oder Gewebe von Interesse geben kann selektiv verwendet werden, um überexprimiert oder knock down Genexpression in diesem begrenzten Bereich, sinkende Nebenwirkungen in den Rest des sich entwickelnden Embryos. In diesem Protokoll beschreiben wir , wie etabliert Xenopus Schicksal Karten verwenden , um die Entwicklung von Xenopus Niere (die pronephros), durch Mikroinjektion in spezifischen Blastomere von 4- und 8-Zellembryos zu zielen. Die Injektion von Lineage Tracer ermöglicht die Verifizierung der spezifischen Ausrichtung der Injektion.Nach Embryonen entwickelt haben, 38 auf die Bühne - 40, ganz-mount Immunfärbung verwendet pronephric Entwicklung sichtbar zu machen, und der Beitrag von Zellen gezielt zu den pronephros beurteilt werden kann. Die gleiche Technik kann andere Gewebetypen angepasst werden, um den pronephros zusätzlich zu zielen.

Mikroinjektionen von 4- und 8-Zell Xenopus - Embryonen mit MEM-RFP mRNA zeigen verschiedene Ebenen zu den pronephros Targeting. 4 zeigt Stufe 40 Embryonen mit korrektem MEM-RFP mRNA Expressionsmuster. Embryos wurden in der linken ventral Blastomere (4A), und sortiert die richtige Expressionsmuster von MEM-RFP mRNA injiziert. Zusätzlich MEM-RFP im proximalen zum Ausdruck zu bringen, Zwischen- und distalen Tubuli der Niere, richtig injizierten Em...

Watch this Scientific Journal Video about Technique to Target Microinjection to the Developing Xenopus Kidney at JoVE.com

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Technik auf Zielmikroinjektions an die Entwicklungs<em&gt; Xenopus</em&gt; Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

The overall goal of this procedure is to target microinjections to the developing xenopus kidney. This method can help address key questions in the field of developmental biology such as how the kidney forms and what genes are important for this process. The main advantage of this technique is that it maximizes the delivery of microinjected reagents to a targeted tissue.Visualization of this method is important because identification of the correct blastomere for targeted microinjection is critical. To begin this procedure both testes were isolated from a single male frog and placed in a 60 millimeter petri dish filled with 10 millileters of testes storage solution. Then store the testes at four degress Celsius.Next, eggs were collected from a female frog in a 100 millimeter petri dish. Pour off excess water from the petri dish. After that cut off a quarter of a testis in Testes Storage Solution.Transfer the piece of testis to the petri dish containing the eggs. Subsequently, cut the testis portion into small pieces. Add MMR plus Gentamycin to the petri dish to cover the eggs.Mix them by swirling the dish and then wait approximately 30 minutes for fertilization to take place at room temperature. After fertilization the pigmented side of the embryos should be facing up. Remove MMR from the petri dish using a transfer pipette and add enough de-jelly solution to cover the embryos.Afterward, swirl the dish over the next few minutes to disolve the jelly coat on the embryos. Once the embryos are closely touching each other in the center of the dish, the jelly coat has been removed. Remove the de-jelly solution with a transfer pipette.Next, wash the de-jellied embryos 3 to 5 times in MMR plus Gentamycin by carefully pouring off the MMR and filling the dish with new MMR. Do not remove all of the MMR from the dish, which may damage the embryos. After that, remove any unfertilized eggs or pieces of testis from the petri dish.To stagger their development, place half of the embryos from a single fertilization in a petri dish kept at 14 degrees Celsius, and the other half of the embryos in a petri dish kept at 18 degrees Celsius. Embryos will develop more slowly at 14 degress Celsius than at 18 degress Celsius. Allowing for multiple sets of injections from a single fertilized clutch.Prepare the injection solution containing point zero one nanogram per nanoliter Membrane-bound Red Fluorescent Protein mRNA. While the embryos are developing to the 4-cell or 8-cell stage. Keep the injection solution on ice until use.Then, load a seven inch replacement glass capillary tube into a needle puller with the top of the replacement tube aligned with the top of the needle puller case. Set the heat number two value to 800 and the pull value to 650 and press the pull button to pull the needle. After that, snip off the tip of the pulled needle with a pair of Dumont forceps.Slip the micropipette collet onto the back of the needle. Next, slip the large hole O-ring onto the back of the needle behind the collet. Now, fill the needle with mineral oil using a 27 gauge hyperdermic needle, being careful not to get air bubbles into the needle.Slip the needle onto the plunger of the microinjector, seating the needle into the large hole of the white plastic spacer installed on the plunger. Secure the needle by tightening the collet. Next, press and hold the Empty"button on the microinjector control box until there are two beeps.Afterward, pipette three microliters of injection solution onto a piece of parafilm. Then, insert the tip of the needle into the bead of injection solution on the parafilm. Press and hold the Fill"button on the microinjector control box to draw the injection solution into the needle.Prior to microinjection, it is important to understand the early cell divisions of the xenopus embryo. The single cell embryo has a darkly pigmented, animal pole and a vegetal pole which is white and yoky. In the two cell embryo the first cleavage typically occurs between the left and right sides of the embryo.These cells contribute equally to the pronephric lineage. The second cleavage divides the dorsal and ventral halves of the embryo leading to a 4-cell embryo. The dorsal cells are smaller and have less pigment than the ventral cells.If injecting into a 4-cell embryo, inject the left ventral blastomere to target the left kidney. The third cleavage bi-sects the animal and vegetal sides. Resulting in an 8-cell embryo.At this point there are four animal blastomeres and four vegetal blastomeres. To target the left kidney of an 8-cell embryo inject into the left ventral vegetal blastomere. Subsequently, fill a 60 millimeter petri dish lined with 500 micron polyester mesh with five percent fycol in MMR plus Gentamycin.Carefully pipette 20 to 30 8-cell embryos into the dish. Using a hair loop manipulate the embryos so that the blastomere to be injected is facing the needle. To target the left kidney, line up the embryos so that the left ventral vegetal blastomeres of 8-cell embryos face the needle.Inject 10 nanoliters of injection solution into the selected blastomere of each embryo in the dish. Then, transfer the injected embryos into the wells of a culture plate with five percent fycol in MMR plus Gentamycin. Incubate the injected embryos at 16 degrees Celsius for approximately one to two hours to allow the injected blastomeres to heal.After that, transfer the microinjected embryos into new wells in the culture plate filled with MMR plus Gentamycin. Incubate the embryos at 14 to 22 degress Celsius until they reach stage 38 to 40. In this procedure transfer 10 to 20 stage 38 to 40 embryos into a glass vial.Add 10 microliters of five percent benzocaine in 100 percent ethanol to the vial. Mix by inverting the vial and wait 10 minutes to anesthetize the embryos. Next, remove MMR from the vial and fill it with MEMFA.Then, place it on a three dimensional, rocking platform for one hour at room temperature. After one hour remove MEMFA from the vial and fill it with 100 percent methanol. Then, place the vial on a three dimensional, rocking platform for ten minutes at room temperature.Repeat this wash step one more time and store the embryos in 100 percent methanol overnight at minus 20 degrees Celsius. Embryos are now fixed and ready for immunostaining. This schematic shows which blastomere to inject for targeting of the left pronephros of xenopus embryos at the 8-cell stage using Membrane-bound Red Fluorescent Protein mRNA as a tracer.Tracer locatlization after injection in the left ventral vegetal blastomere is shown in red while the kidney is labeled in green. This image shows that the kidney was stained green with antibodies 3G8 and 4A6. Here is a close up view of the kidney region showing tracer loacalization and here is the merged image showing the co-localization of the tracer with the kidney.Shown here is the confocal image of the kidney of a second embryo stained with 3G8 and 4A6. This is the localization of the red tracer in the kidney and surrounding tissue and this is the merged image showing the co-localization of the tracer 3G8 and 4A6 in the kidney. Prior to performing this procedure it's important to become familiar with the early xenopus embryo.This will allow you to properly target microinjections through the established xenopus fade map. Once mastered, this technique can be adapted to target other tissues such as the neural tube or heart.

technique-to-target-microinjection-to-the-developing-xenopus-kidney

Research

JoVE Journal

Developmental Biology

Technique to Target Microinjection to the Developing Xenopus Kidney

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Manipulation und<em&gt; In-vitro-</em&gt; Ausbau der<em&gt; Xenopus laevis</em&gt; Eizellen, von Intrazytoplasmatische Spermieninjektion gefolgt, um embryonale Entwicklungsstudie

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Forschung

Entwicklungsbiologie

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

Mittels konfokaler Analyse<em&gt; Xenopus laevis</em&gt; Um Modulatoren der Wnt und Shh Morphogen Gradienten Untersuchen

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Stammzell-ähnlichen<em&gt; Xenopus</em&gt; Embryonale Explantate bis Früh Neurale Entwicklungs- Eigenschaften Studieren<em&gt; In-vitro-</em&gt; Und<em&gt; In-vivo-</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

Ein 5-mC-Dot-Blot-Assay, der den DNA-Methylierungsgrad der Chondrozyten-Dedifferenzierung quantifiziert<em&gt; In vitro</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Präziser zellulärer Ablationsansatz zur Modellierung akuter Nierenverletzungen bei der Entwicklung von Zebrafisch

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

Erzeugung von genetisch modifizierten Mäusen durch die Mikroinjektion von Oozyten

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

Bewertung von oxidativen Schäden in den primären Maus-Augen-Oberflächenzellen/Stammzellen als Reaktion auf Uv-C-Schäden (UV-C)

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Mikroinjektion von DNA in Augenknospen in Xenopus laevis Embryonen und Bildgebung von GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Ganz-Mount-In-Situ-Hybridisierung in Zebrafisch-Embryonen und Tube-Formations-Assay in iPSC-ECs zur Untersuchung der Rolle von Endoglin in der Gefäßentwicklung

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

Isolation, Kultur und adipogene Induktion von Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...