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><tbody><tr><td>Pregnant Mare Serum Gonadotropin (PMSG)&amp;nbsp;</td><td>MSD Animal Health</td><td>Vm 01708/4309</td><td>PMSG-Intervet 5000iu powder and solvent for solution for injection</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt (MS222)</td><td>Sigma</td><td>A5040</td><td>For anesthesia</td></tr><tr><td>Liberase TM Research Grade</td><td>Roche</td><td>05 401 127 001</td><td>For oocyte defolliculation. Store at -20&amp;nbsp;&amp;#176;C.</td></tr><tr><td>Drummond Nanoject</td><td>Drummond Scientific Company</td><td>3-000-205/206</td><td>For microinjection</td></tr><tr><td>glass capillary&amp;nbsp;</td><td>Alpha laboratories</td><td>7&amp;rdquo; Drummond #3-000-203-G/XL</td><td>For microinjection</td></tr><tr><td>Micropipette Puller&amp;nbsp;</td><td>SUTTER Instrument</td><td>Model D-97</td><td>For microinjection</td></tr><tr><td>UltraPure Agarose</td><td>Invitrogen</td><td>16500-500</td><td>For &lt;em&gt;in vitro&lt;/em&gt; maturation and ICSI</td></tr><tr><td>14 ml ultra-clear centrifuge tube&amp;nbsp;</td><td>Beckman Coulter United Kingdom Ltd</td><td>344060</td><td>For sperm purification</td></tr><tr><td>OptiPrep Density Gradient Medium (Iodixanol)</td><td>Sigma</td><td>D1556</td><td>For sperm purification</td></tr><tr><td>Digitonin</td><td>Sigma</td><td>D141</td><td>Cell permeabilization reagent</td></tr><tr><td>Shaker</td><td>Hybaid&amp;nbsp;</td><td>HB-SHK-1</td><td>For oocyte defolliculation</td></tr><tr><td>Dissecting microscope (Stereo zoom microscope)</td><td>ZEISS</td><td>Semi SV6</td><td>For oocyte collection and microinjection</td></tr><tr><td>50 &amp;mu;m pore filter</td><td>CellTrics</td><td>04-0042-2317</td><td>For sperm purification</td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima L-100XP</td><td>For sperm purification</td></tr><tr><td>50 ml centrifuge tube</td><td>Cellstar Greiner Bio-One</td><td>227261 or 210261</td><td>Oocyte collection and defolliculation reaction</td></tr><tr><td>15 ml centrifuge tube</td><td>FALCON</td><td>352097</td><td>For sperm purification</td></tr><tr><td>90 mm Petri dish&amp;nbsp;</td><td>Thermo Scientific</td><td>101VR20/C</td><td></td></tr><tr><td>Easy-Grip Cell Culture Dish, 60&amp;nbsp;x&amp;nbsp;15 mm</td><td>FALCON</td><td>353004</td><td></td></tr><tr><td>Easy-Grip Cell Culture Dish, 35&amp;nbsp;x&amp;nbsp;15 mm</td><td>FALCON</td><td>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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

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

22.4K 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

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 ...

Gelişim Biyolojisi

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, ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Bir kullanılması Fonksiyonel Klonlama<em&gt; Xenopus</em&gt; Oosit ekspresyon sistemi

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

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 ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Xenopus laevis Oosit, Mikroenjeksiyon

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Biyoloji

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Gübrelenmesi Xenopus oositleri Host Transfer Yöntemi kullanılarak

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Canlı hücre Görüntüleme ve Kantitatif Analiz Embriyonik Epitel Hücreleri Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Transgenik, üretimi Xenopus laevis Kısıtlama Enzim aracılığı ile Entegrasyon ve Nükleer Nakli

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Manipülasyon ve

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Konfokal Analizi Kullanımı

Kök hücre benzeri

Bir kullanılması Fonksiyonel Klonlama

Gelişmekte olan mikroenjeksiyon Hedef Tekniği

Kullanılması Anne Gen Ürünlerinin Fonksiyonel Manipülasyonu

Embriyonik Anjiyojenezin Görselleştirilmesi ve Nicel Analizi

Xenopus laevis Oosit, Mikroenjeksiyon

Gübrelenmesi Xenopus oositleri Host Transfer Yöntemi kullanılarak

Canlı hücre Görüntüleme ve Kantitatif Analiz Embriyonik Epitel Hücreleri Xenopus laevis

Transgenik, üretimi Xenopus laevis Kısıtlama Enzim aracılığı ile Entegrasyon ve Nükleer Nakli