We are grateful to Gurdon laboratory members for useful discussion. K.M. is a Research Fellow at Wolfson College and is supported by the Herchel Smith Postdoctoral Fellowship and the Great Britain Sasakawa Foundation. Gurdon laboratory is supported by grants from the Wellcome Trust (RG69899) and MRC to J.B.G.

<ol>
	<li>Miyamoto, K., Gurdon, J. B., Trounson, A., Gosden, R., Eichenlaub-Ritter, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+amphibian+egg+to+understand+the+mammalian+oocyte.">Insights into the amphibian egg to understand the mammalian oocyte.</a> Biology and Pathology of the Oocyte: Role in Fertility, Medicine, and Nuclear Reprogramming. , 1-11 (2013).</li><li>Heasman, J., Kofron, M., Wylie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-catenin+signaling+activity+dissected+in+the+early+Xenopus+embryo:+a+novel+antisense+approach.">Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach.</a> Developmental biology. 222 (1), 124-134 (2000).</li><li>Oelgeschlager, M., Kuroda, H., Reversade, B., De Robertis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chordin+is+required+for+the+Spemann+organizer+transplantation+phenomenon+in+Xenopus+embryos.">Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos.</a> Developmental cell. 4 (2), 219-230 (2003).</li><li>Szenker, E., Lacoste, N., Almouzni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+requirement+for+HIRA-dependent+H3.3+deposition+revealed+at+gastrulation+in+Xenopus.">A developmental requirement for HIRA-dependent H3.3 deposition revealed at gastrulation in Xenopus.</a> Cell reports. 1 (6), 730-740 (2012).</li><li>Heasman, J., Holwill, S., Wylie, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+of+cultured+Xenopus+oocytes+and+use+in+studies+of+maternally+inherited+molecules.">Fertilization of cultured Xenopus oocytes and use in studies of maternally inherited molecules.</a> Methods in cell biology. 36, 213-230 (1991).</li><li>Heasman, J., 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=Overexpression+of+cadherins+and+underexpression+of+beta-catenin+inhibit+dorsal+mesoderm+induction+in+early+Xenopus+embryos.">Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos.</a> Cell. 79 (5), 791-803 (1994).</li><li>Hulstrand, A. M., Schneider, P. N., Houston, 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=The+use+of+antisense+oligonucleotides+in+Xenopus+oocytes.">The use of antisense oligonucleotides in Xenopus oocytes.</a> Methods. 51 (1), 75-81 (2010).</li><li>Miyamoto, 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=Nuclear+Wave1+is+required+for+reprogramming+transcription+in+oocytes+and+for+normal+development.">Nuclear Wave1 is required for reprogramming transcription in oocytes and for normal development.</a> Science. 341 (6149), 1002-1005 (2013).</li><li>Halley-Stott, R. 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=Mammalian+nuclear+transplantation+to+Germinal+Vesicle+stage+Xenopus+oocytes+-+a+method+for+quantitative+transcriptional+reprogramming.">Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes - a method for quantitative transcriptional reprogramming.</a> Methods. 51 (1), 56-65 (2010).</li><li>Astrand, C., Belikov, S., Wrange, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+characterizes+chromatin+presetting+by+NF1+and+Oct1+and+enhances+glucocorticoid+receptor+binding+to+the+MMTV+promoter.">Histone acetylation characterizes chromatin presetting by NF1 and Oct1 and enhances glucocorticoid receptor binding to the MMTV promoter.</a> Experimental cell research. 315 (15), 2604-2615 (2009).</li><li>Amaya, E., Kroll, K. 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+method+for+generating+transgenic+frog+embryos.">A method for generating transgenic frog embryos.</a> Methods in molecular biology. 97, 393-414 (1999).</li><li>Kroll, K. L., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+Xenopus+embryos+from+sperm+nuclear+transplantations+reveal+FGF+signaling+requirements+during+gastrulation.">Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation.</a> Development. 122 (10), 3173-3183 (1996).</li><li>Smith, S. J., Fairclough, L., Latinkic, B. V., Sparrow, D. B., Mohun, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+transgenesis+by+sperm+nuclear+injection.">Xenopus laevis transgenesis by sperm nuclear injection.</a> Nature protocols. 1 (5), 2195-2203 (2006).</li><li>Drury, K. C., Schorderet-Slatkine, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cycloheximide+on+the+'autocatalytic'+nature+of+the+maturation+promoting+factor+(MPF)+in+oocytes+of+Xenopus+laevis.">Effects of cycloheximide on the 'autocatalytic' nature of the maturation promoting factor (MPF) in oocytes of Xenopus laevis.</a> Cell. 4 (3), 269-274 (1975).</li><li>Smith, L. D., Ecker, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+oocyte+nucleus+in+physiological+maturation+in+Rana+pipiens.">Role of the oocyte nucleus in physiological maturation in Rana pipiens.</a> Developmental biology. 19 (3), 281-309 (1969).</li><li>Miyamoto, K., Pasque, V., Jullien, J., Gurdon, 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=Nuclear+actin+polymerization+is+required+for+transcriptional+reprogramming+of+Oct4+by+oocytes.">Nuclear actin polymerization is required for transcriptional reprogramming of Oct4 by oocytes.</a> Genes &amp; development. 25 (9), 946-958 (2011).</li><li>Schneider, P. N., Hulstrand, A. M., Houston, 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=Fertilization+of+Xenopus+oocytes+using+the+host+transfer+method.">Fertilization of Xenopus oocytes using the host transfer method.</a> J. Vis. Exp. (45), (2010).</li><li>Ishibashi, S., Cliffe, R., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+bi-allelic+mutation+rates+using+TALENs+in+Xenopus+tropicalis).">Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis).</a> Biology open. 1 (12), 1273-1276 (2012).</li><li>Sakane, Y., 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=Targeted+mutagenesis+of+multiple+and+paralogous+genes+in+Xenopus+laevis+using+two+pairs+of+transcription+activator-like+effector+nucleases.">Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases.</a> Development, growth &amp; differentiation. 56 (1), 108-114 (2014).</li><li>Nakayama, T., 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=Simple+and+efficient+CRISPR/Cas9-mediated+targeted+mutagenesis+in+Xenopus+tropicalis.">Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.</a> Genesis. 51 (12), 835-843 (2013).</li><li>Guo, X., 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=Efficient+RNA/Cas9-mediated+genome+editing+in+Xenopus+tropicalis.">Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.</a> Development. 141 (3), 707-714 (2014).</li></ol>

The authors have nothing to disclose.

Biz burada detay anne faktörleri tüketmek ya da döllenme öncesi dışsal faktörleri aşırı ifade için yeni bir yöntem. Bu sistem iki microinjections gerektirir, ancak bunun yerine ev sahibi transferi yöntemi 7,17 kullanılan kurbağa ameliyatı, atlar. Bu hayvan bakımı açısından kurbağa ameliyatı adımı kaldırmak için en uygunudur. Dahası, biz deneylerin başarısını etkileyen bir faktör biyolojik kurtulmak anlamına transfer deneyleri için ev sahibi kadın kurbağa kalitesi dikkate almak zorunda değilsiniz. Bu nedenle, bu sistemde PMSG-prime kurbağa elde edilen oosit kalitesi başarılı deneyler için anahtar önemli bir biyolojik faktördür. oosit kalitesi yumurtalık veya sonrasında defolliculation toplandıktan sonra yargılanabilir. Herhangi bir anormallik bu aşamada görülürse, yeni bir kurbağa başka yumurtalık toplamak için en uygunudur. Eğer oosit kalitesini kontrol edebilirsiniz başka nokta oosit olgunlaşması peşinde. Eğer progesteron az% 80E-tedavi oosit olgunlaşma belirtileri gösteriyor, daha fazla analiz için embriyo yeterli sayıda elde etmek mümkün olmayabilir. enzimatik defolliculation yerine kılavuzu defolliculation kullanımı da defolliculation için gerekli emek azaltmak için bu sistemi kullanan bir başka avantajdır. Ancak, manuel defolliculation enzimatik tedaviden daha iyi bir gelişme verebilir ki hala mümkün. Şekil 3&#39;te gösterildiği gibi, in vitro olarak yaklaşık% 10, yüzme larva dönemini ulaşabilir oosit olgunlaşması. Bu veriler, 8 ve yeni enjeksiyon deneyleri rapor da dahil olmak üzere 13 bağımsız deneyden toplanan. Sunucu aktarma yöntemi transfer oosit% 30-60 neurula aşamasına 17 ulaşabilirsiniz beri anlamlı veri elde etmek için her tedavi 75-150 oosit ihtiyacı var. Önerilen yöntem, normal bölünen embriyolar, yaklaşık% 40-60 ulaşabildiklerinden her bir deney grubu 200-300 oosit ile başlarkas tepki aşaması. Bu veriler her iki yöntem de makul bir gelişim hızı desteklediğini gösteriyor. Biz şimdiye kadar bu yaklaşım metamorfoz ile gelişimini destekler belirten, bu tekniği kullanarak kontrol mRNA enjekte ve kontrol antisens oligo-enjekte oosit 10 canlı kurbağa almış. Bizim oosit manipülasyon-ICSI yöntemi yakında döllenmeden sonra, çok erken embriyonik gelişimi sırasında maternal faktörlerin rolünü test etmek için bir fırsat sağlar. Buna ek olarak, gübreleme öncesi kromatin değiştirici faktörlerin ekspresyonu mümkün gelişimi için gerekli olan anne kromatin durumlarını anlamak için gübreleme öncesi anne kromatin pişmanlık yapabilir. Bu strateji, aynı zamanda bu bile gübreleme önce ifade edilebilir beri Transkripsiyon aktivatörü gibi efektör nükleaz (TALEN) 18,19 ve CRISPR / CAS9 20,21 olarak mevcut gen düzenleme sistemleri ile de çalışabilir. Bu nedenle, yeni bir yaklaşım potenti vardırark gelecek bir çok uygulama için kullanılır.

Xenopus laevis geliştirme 1 incelemek için yaygın olarak kullanılan, güçlü bir model organizmadır. Xenopus yumurta ve bol (memeli meslektaşları karşılaştırma yaklaşık 0.1 mm olmak, yaklaşık 1.2-1.4 mm) alışılmadık büyük olmasıdır. Yumurta (4,000-8,000 hücreleri ile sahnede 8-8,5 meydana gelen, MBT) orta-blastula geçiş kadar embriyo gelişimini götürmek için yeterli olan anne sentezlenmiş ve depolanan bileşenler içerir. MBT sonra daha da geliştirilmesi doğrudan embriyonik gen ürünleri üreten zigotik genom aktivasyonu, eşlik ediyor. Çeşitli çalışmalar gelişimi için önemli anne faktörlerini belirlemeyi amaçladık. Birçok çalışma, anne proteinlerin durum bozunma gastrula aşamasında 2-4 de gözlenebildiği döllenmiş embriyolar haline morfolino oligonükleotitler de dahil olmak üzere anti-his oligonükleotidler (oligos) enjeksiyonu dayanır. Alternatif olarak, mRNAlar döllenmiş em enjekte edilirbryos gen fonksiyonlarını rahatsız veya aşın proteinlerin kaderlerini izlemek için. Bununla birlikte, tek bir hücre aşamasında embriyoların içine enjeksiyon normal MBT önce çok erken evrelerinde maternal protein ifade seviyelerini etkilemez. Heasman ve Wylie, bu sorunu aşmak için 5 konak aktarma yöntemi kurdu. Bunların bir yöntemde, el defolike oosit antisens oligoları ile enjekte edilir ve ana kadın 6 transfer edilir. Erken dönemde embriyo gelişimi olarak azaldı proteinlerin rolleri incelenmiş böylece proteinler Fertilizasyondan önce aşağı regüle edilir. 7 &#39;da gözden geçirilen bu anne tüketme yöntemi olup, anne proteinlerin çeşitli benzersiz gelişim roller belirlenmesine yol açmıştır. Döllenme önce mRNA anne tükenmesi ya da aşırı ifadesi manuel defolliculation ve ev sahibi transferi olmadan elde edildiği, bu raporda, biz ayrıntı bizim yeni geliştirilen yöntem, içinde8. Manuel defolliculation zaman ve konak transferi bir sürü genellikle böylece anne tükenme yöntemi sık kullanımı engelleyici, usta teknikleri ve hayvan cerrahisi için özel lisans ihtiyacı gerektirir. Defolliculation ve ev sahibi transferi sırasıyla, enzimatik defolliculation 9,10 ve intrasitoplazmik sperm enjeksiyonu (ICSI) 11-13 ile ikame edilir. Yumurta ICSI başlangıçta transgenik kurbağalar 12 üretmek için kullanıldı. Sperm ve embriyonik çekirdekleri ayrıca in vitro olgunlaşması amfibi oosit 11,14,15 nakledildi. Burada, biz in vitro olgunlaştı oosit sperm enjekte etmek bizim adım adım yöntemi göstermek antisens oligolar veya mRNA ile ön-enjekte.

<table border="0" cellpadding="0" cellspacing="0" width="589">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:87px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pregnant Mare Serum Gonadotropin (PMSG)&#160;</td>
			<td style="width:87px;">MSD Animal Health</td>
			<td style="width:119px;">Vm 01708/4309</td>
			<td>PMSG-Intervet 5000iu powder and solvent for solution for injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethyl 3-aminobenzoate methanesulfonate salt (MS222)</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">A5040</td>
			<td>For anesthesia</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Liberase TM Research Grade</td>
			<td style="width:87px;">Roche</td>
			<td style="width:119px;">05 401 127 001</td>
			<td>For oocyte defolliculation. Store at -20oC</td>
		</tr>
		<tr height="72">
			<td height="72" style="height:72px;width:216px;">Drummond Nanoject</td>
			<td style="width:87px;">Drummond Scientific Company</td>
			<td style="width:119px;">3-000-205/206</td>
			<td>For microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">glass capillary&#160;</td>
			<td style="width:87px;">Alpha laboratories</td>
			<td style="width:119px;">7&#8221; Drummond #3-000-203-G/XL</td>
			<td>For microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette Puller&#160;</td>
			<td style="width:87px;">SUTTER Instrument</td>
			<td style="width:119px;">Model D-97</td>
			<td>For microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraPure Agarose</td>
			<td style="width:87px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td>For invitro maturation and ICSI</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">14 ml ultra-clear centrifuge tube&#160;</td>
			<td style="width:87px;">Beckman Coulter United Kingdom Ltd</td>
			<td style="width:119px;">344060</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">OptiPrep Density Gradient Medium (Iodixanol)</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">D1556</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digitonin</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">D141</td>
			<td>Cell permeabilization reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shaker</td>
			<td style="width:87px;">Hybaid&#160;</td>
			<td style="width:119px;">HB-SHK-1</td>
			<td>For oocyte defolliculation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscope (Stereo zoom microscope)</td>
			<td style="width:87px;">ZEISS</td>
			<td style="width:119px;">Semi SV6</td>
			<td>For oocyte collection and microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 &#956;m pore filter</td>
			<td style="width:87px;">CellTrics</td>
			<td style="width:119px;">04-0042-2317</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ultracentrifuge</td>
			<td style="width:87px;">Beckman Coulter</td>
			<td style="width:119px;">Optima L-100XP</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50 ml centrifuge tube</td>
			<td style="width:87px;">Cellstar Greiner Bio-One</td>
			<td style="width:119px;">227261 or 210261</td>
			<td>Oocyte collection and defolliculation reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml centrifuge tube</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">352097</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">90 mm Petri dish&#160;</td>
			<td style="width:87px;">Thermo Scientific</td>
			<td style="width:119px;">101VR20/C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easy-Grip Cell Culture Dish, 60x15 mm</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">353004</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easy-Grip Cell Culture Dish, 35x15 mm</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">351008</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In vitro olgunlaştı oosit kullanarak ICSI embriyoların Embriyonik gelişim (Şekil 3A) incelendi. MII sahneye GV oosit olgunlaşması oranları değişkendir ve büyük ölçüde oosit kalitesine bağlıdır. İyi deneylerde, GV oosit neredeyse% 100 nihayet MII aşamada yumurta haline, oosit olgunlaşması progesteron ve gösteri işaretleri cevap. Tüm oositler ICSI olgunlaşmış tabi tutulur ve enjekte klivaj edilen yumurtaların yaklaşık% 25&#39;i (Şekil 3A, n = kontrolü için 13 oligo enjekte oosit karıştırılmış ve n = herhangi bir oligo-enjekte edilmiş oositlerde 7) yapılmıştır. Yaklaşık% 60 ya da kontrol oligo-enjekte edilmiş oositlerde veya enjeksiyon oosit elde ayrılır embriyolar,% 80, sırasıyla Blastula / gastrula aşamasına. Blastula / gastrula embriyoların% 82 n iyi kalitede olduğunu ise blastula / gastrula embriyolar arasında, oligo-enjekte örneklerde embriyoların yaklaşık yarısı (anormal bölünme ve apoptoz neredeyse hiçbir işaret) iyi kalitedeile enjekte örnekleri (Şekil 3A). Enjekte örnekleri içinde% 60 ve parçalanan embriyoların% 29 (Şekil 3A) yaptı ise Son olarak,% 41 ve oligo enjekte örneklerde bölünen embriyolar% 11, sırasıyla, kas tepkisini ve yüzme larva aşamalarında ulaşmıştır. Bu ICSI embriyolar, normal ve anormal embriyolar (Şekil 3B) karışımlarıdır. Bazıları metamorfoz geçiren ve kurbağalar 8 olgun geliştirmek. Bu sonuçlar IVM ve ICSI takip GV oosit içine antisens oligonükleotitlerin, enjeksiyonu, etkili erken embriyo gelişimini sağlayan öneririz. Antisens oligos kendisi enjeksiyon embriyonik gelişimini azaltır rağmen, biz hala böyle gelişimsel oranları kontrol gibi birçok deneysel amaçlar için yeterli embriyolar elde edebiliyoruz, RT-PCR, böylece batı leke ve. <img alt="Şekil 1," src="/files/ftp_upload/52496/52496fig1highres.jpg" /> İncir<html> ure 1: Xenopus tipik örnekleri, in vitro maturasyon deneyler için kaliteli ya da kötü kalite oosit laevis. (A) kötü kaliteli oosit bir örnek. Örneğin, yamalı pigmentasyon gösteren oositlerin daha sonraki deneyler için kullanılmamaktadır. Her Xenopus oosit çapı yaklaşık 1.2-1.4 mm laevis. (B) kısmen defolike oosit. oosit sağ yarısı kan damarlarının varlığı ile ayırt edilebilir folikül hücreleri, ile kaplıdır. Bir ok folikül hücreleri serbest ve enjeksiyon iğnesi enjekte edildiği içine bir alanı gösterir. (C) eşit büyüklükte ve hayvan yarımkürede ve bitkisel yarımkürede arasında açık kontrast eşit pigmentli hayvan hemisfer göstermek kaliteli oosit, bir örnek. 2496 / 52496fig2highres.jpg &quot;/&gt; Şekil 2:. Xenopus öncesi ve olgunlaşma sonrası oosit laevis oosit olgunlaşması sonra, berrak beyaz lekeler hayvan hemisfer ve folikül hücreleri üst kısmında görünen soyulmuş edilir. Her bir Xenopus laevis oosit çapı yaklaşık 1 mm&#39;dir. <img alt="Şekil 3," src="/files/ftp_upload/52496/52496fig3highres.jpg" /> Şekil 3: in vitro olgunlaştı oosit üretilen ICSI embriyoların, geliştirilmesi. Her aşamada ICSI embriyolar (A) Development özetlenmiştir. Kontrol IVM ve ICSI takip antisens oligonükleotidler (kontrol oligo enjeksiyon) veya enjeksiyon (non enjeksiyon) olmadan, şifreli olan oositler enjekte edildi. Her aşamada embriyolar gelen sayı çubukları üzerinde gösterilir. ± SEM gösterilir idi. N = 4-13 bağımsız deney / farklı females. (B) ICSI embriyoların hayatta örnekleri. Bu tailbud aşamasındaki embriyolar yaklaşık 200 oosit, bir başlangıç ​​materyali olarak kullanıldığı bir deneyde, imal edilmiştir.

Watch this Scientific Journal Video about Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development at JoVE.com

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.

Manipülasyon ve<em&gt; İn Vitro</emVe&gt; Olgunlaşma<em&gt; Xenopus laevis</emIntrasitoplazmik sperm enjeksiyonu izledi&gt; oositler, Embriyonik Geliştirme Çalışması

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

manipulation-vitro-maturation-xenopus-laevis-oocytes-followed

Research

JoVE Journal

Developmental Biology

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

22.2K Görüntüleme.  University of Cambridge. 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.

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

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

Floresan protein ifadesindeki<em&gt; Branchiostoma lanceolatum</em&gt; Döllenmemiş Oositlerinin içine mRNA Enjeksiyon

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Gelişim Biyolojisi

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Elektroporasyon tarafından Embriyonik Fare Koklear Eksplantlarından Kültür ve Gen Transferi

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

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.

Konfokal Analizi Kullanımı<em&gt; Xenopus laevis</em&gt; Wnt ve Shh morfogen Gradyanların modülyatorlar Soruşturma

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.

Kök hücre benzeri<em&gt; Xenopus</em&gt; Embriyonik Eksplantlarındaki Erken Nöral Gelişim Özellikleri Okuyacak<em&gt; İn Vitro</em&gt; Ve<em&gt; İn Vivo</em

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

Technique to Target Microinjection to the Developing Xenopus 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 embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

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.

Gelişmekte olan mikroenjeksiyon Hedef Tekniği<em&gt; Xenopus</em&gt; Böbrek

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 ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Kullanılması Anne Gen Ürünlerinin Fonksiyonel Manipülasyonu<em&gt; İn Vitro</emZebra balığı&gt; Oosit Olgunlaştırma

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Embriyonik Anjiyojenezin Görselleştirilmesi ve Nicel Analizi<emXenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Embriyonik tavuk doku gelişimi çalışmaya Organotypic kültür yöntemi

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

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.

Xenopus laevis Embriyolarında Göz tomurcuklarına DNA'nın mikroenjeksiyonu ve GFP'nin Sağlam Optik Aksonal Arbors'u İfade Etme Görüntülemesi, Yaşayan Xenopus Kurbağaları

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

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Xenopus laevis'te Nöronal Öncül Karakterizasyonu için Floresan Kalsiyum Görüntüleme ve Daha Sonra Yerinde Hibridizasyon

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...