This work was supported by a Professional Development Grant to C.Z.P. from the Shepherd University Foundation. The authors wish to thank Brett Zirkle and Malia Deshotel for helpful discussions on the protocols, and Dr. Carol Hurney for generous assistance.

Tüm deney prosedürleri Virginia Kurumsal Hayvan Bakım ve Kullanım Kurulu Üniversitesi tarafından onaylanmıştır. Not: Şekil 1, deneysel prosedürler şematik görünümünü gösterir. Oosit hazırlanması 1. <ol><li> Ön asal X oosit izolasyon önceden Gebe Kısrak Serum Gonadotropin 150 U (PMSG) yaklaşık bir hafta ile kadın laevis. 29 G iğne ile 1 cc steril şırınga ile dorsal lenf kese içine 1 ml 150 U / ml PMSG enjekte edilir. </li><li> Oosit enjeksiyonu ve oosit-hayvan kap deneyi için çözümler hazırlayın. <ol><li> Ca Hazırlama ++ / Mg ++ içermeyen OR2 (OR2-): 82 mM NaCI, 2.5 mM KCI, 1.5 mM Na 2 HPO 4, 50 mM HEPES pH 7.2. </li><li> OR2- artı 1 mM CaCl2 ve 1 mM MgCl2: ya da 2&#39;yi hazırlayın. </li><li> % 60 Leibovitz L15, 0.4 mg / ml BSA, 100 ug / ml gentamisin, pH 7,8: OCM hazırlayın. </li><li> Ön1x MBS hazırlanması: 88 mM NaCI, 1 mM KCI, 0.7 mM CaCl2, 1 mM MgSO 4, 5 mM HEPES (pH 7.8), 2.5 mM 3 NaHCO. </li><li> 1x MBS% 3 Ficoll hazırlayın. </li><li> 1x NAM hazırlayın: 110 mM NaCI, 2 mM KCI, 1 mM Ca (NO 3) 2, 1 mM MgSO 4, 0.1 mM EDTA, 1 mM NaHCO 3, 2 mM Na 3 PO 4, pH 7.4 ile yıkanır. </li></ol></li><li> 0.1X MBS Etil 3-aminobenzoat metansülfonat tuzunun% 0.03 çözeltisi (MS222) &#39;de kadın anestezisi için çözelti kurbağa yerleştirerek (ilk 10 ml% 95 etanol içinde 0.3 g MS222 çözülür, daha sonra 1 L, 0.1X MBS sulandırmak) 10-15 dakika veya tepkisiz kadar. <ol><li> Bu anestezi yanıt vermiyor sağlamak (eksik anestezi kurbağa uzuvları taşımak veya bedeni teslim) onun sırtına anestezi kurbağa çevirerek tam olup olmadığını kontrol edin. </li></ol></li><li> Cerrahi forseps ile yumurtalık dokusu izole bisturi cilt ve vücut duvarı boyunca abdominal insizyon yapılıp, yumurtalık fragmanlarını izoleve makas. OR2- içine yumurtalık parçaları yerleştirin. 24 mm&#39;lik kavisli iğne 3-0 ipek sütür ile vücut duvarı kapatın ve aynı sütür ile ayrı ayrı cilt kapatın. Su içinde 1 g / L akvaryum tuzu kadın kurtarma izin verir. </li><li> (- 20 oosit 10) adet ve taze OR2- transfer küçük içine ince forseps, gözyaşı yumurtalık dokusu kullanma. </li><li> 1 saat boyunca karıştırma cihazı üzerinde, hafifçe ajite taze kolajenaz geçiş ve bir saat daha çalkalayarak OR2- 2.0 mg / ml kollajenaz A Defolliculate yumurtalık fragmanları. </li><li> Yıkama küçük oosit atarak OR2 [Ca ++ / Mg ++ ihtiva eden] 10 kez, oositlerde. </li><li> OCM iki kez oosit yıkayın ve taze OCM transfer. Görsel inceleme üzerine boyutuna göre Evre VI oosit izole edin ve olgunlaşmamış (küçük) oosit atın: Sahne VI oositlerin hayvan yarımkürede bile pigmentasyon ile olgunlaşmamış oositlerin büyüktür ve çapı yaklaşık 1.2-1.4 mm. </li><li> 20 ° C i - 18 oosit koruyunenjeksiyondan önce n OCM. Not: Agaroz kaplı petri kaplarına çanak oositlerin yapışmasını en aza indirmek için de kullanılabilir. </li></ol> Kütüphane Transkriptler 2. Enjeksiyon <ol><li> Gelişme uygun bir aşamada (örneğin, nöral plak aşaması 14 10) ekstre yönlü bir cDNA kütüphanesi 15 kullanarak, RNA yı hazırlamak. Bir cDNA kütüphanesi satın alın ya da aşağıda dört adımda genel başına bir ticari kit kullanılarak bir inşa. <ol><li> Poli (A) + RNA (Malzeme Tablo bakınız) için zenginleştirmek için bir ürünü kullanırken ve üreticinin yönergeleri izleyerek yaklaşık 5 ug mRNA izole edin. Not: transkript (istenilen doku mikrodiseksiyon sonra, nöral levha gibi) bir indükleme dokuya sınırlı olabilir cDNA kütüphanesini üretmek için kullanılır, ya da belirli bir aşamada bütün embriyolardan olabilir. </li><li> Üreticinin yönergeleri izleyerek bir ticari kit ile cDNA üretin. </li><li> Bir Vecto içine cDNA ng, yaklaşık 20 Arterr ticari kit veya uygun başka bir vektör ile birlikte. Uygun bir plazmid vektörü mRNA stabilitesi gibi 5 &#39;β-globin ve 3 poli (A) sekansları (PCS2 +, pTnT, pCS105) için dizileri içerir. </li><li> Isı şok dönüşümü için tedarikçinin önerdiği protokol kullanılarak yetkili bakteri hücrelerine ligasyonu Transform. Not: Alternatif olarak, açık okuma çerçevesi (ORFeome) klonları koleksiyonu, ticari olarak mevcuttur ve enjeksiyon için transkriptleri oluşturmak için kullanılabilir. </li></ol></li><li> 10 4 karmaşıklık (10 Nisan - 10 Mayıs toplam karmaşıklığı) 10 3 Plazmidlerin 10 havuzları hazırlayın. 10.000 kolonilerin her - Bu 1000 ile 10 plakaları temsil eder. <ol><li> 37 ˚C de 18 saat ve 7 ml LB bir cam dağıtıcı ile hafif basınç her gelen koloniler toplamak - Plaka kütüphane kültürü 10 15 cm LB-ampisilin plakalar üzerine, 12 büyüyecek </li><li> 0.5 ml bir gliserol stok hazırlayın (-20 &amp; 0.2 ml steril gliserol ve mağaza eklemek# 730 C) ve bir standart ile, üretici firmanın talimatlarına aşağıdaki ticari olarak temin edilebilir DNA miniprep kiti DNA hazırlamak için, kalan 6.5 ml kullanılır. </li></ol></li><li> 1.5 saat - Enzim 1 için 37 ° C&#39;da 16 sindirimi, uygun kısıtlama - (2,0 ug 1,0) havuzlanmış plazmid DNA Linearize. Kullanılan RNA polimeraz kit özelliklerine göre su içinde etanol çökeltme ve yeniden süspansiyon haline getirildi, fenol / kloroform ekstraksiyonu ile linearize DNA izole edin. Üreticinin yönergeleri izleyerek bir ticari RNA polimeraz kiti ile duyu RNA sentez. </li><li> Yaklaşık 20 mikron çapında (cam kılcal boru ile bir iğne çektirmenin kullanarak) mikroenjeksiyon için iğne hazırlayın; kalibre oküler mikrometre ile bir bileşik mikroskop iğne uçları ölçün. Not: İğne çektirmesi ayarları ince forseps ile kırıldığı zaman istenilen uç boyutu ortaya çıkarır şekilde ince uçlu bir iğne üretmek ampirik olarak tespit edilmelidir. </li><li> (Içine kil itin hazırlayınçanak alt) üzerine muntazam tabakalı kil astarlı 35 x 10 mm mikroenjeksiyon sırasında yerinde oosit tutmak için paralel oluklar petri yemekler; alev ucunda erimiş bir alışveriş merkezi probu veya Pasteur pipeti ile oluklar üretirler. Kil, bir alternatif olarak, bir elastomer kalıp 17, girinti yaparak agaroz yemekler hazırlamak. Kil astarlı yemekleri satırları 1X MBS% 3 Ficoll (yaklaşık 2 ml) geniş çaplı bir pipet ile Aktarım oosit. </li><li> Mikroenjektör kullanarak, 1 ng / nl RNA ile iğne doldurup (oosit sitoplazması kadar çizim önlemek için) hafif pozitif basınç üretmek için dengesini ayarlamak. </li><li> Ekvator bölgesinde yaklaşık 20 nl RNA ile oosit enjekte edilir. Daha sonra, Ficoll-MBS kalır 1x MBS yavaşça aktarmak için enjekte edilmiş oositlerde için 1 saat izin verin. Önceden hayvan başlık tahlili için 20 ° C&#39;de 24 saat - 8 inkübe edin. </li></ol> 3. Hayvan Cap Testi <ol><li> Ince forseps, saç döngü, kavisli lamel parçaları, kil astarlı dis 3 / 4x NAM hazırlayın ve eldefincan şekilli girinti çıkıntılar ile hes oosit tutun. Kavisli parça üreten, (4 mm - - 2 mm x 2 yaklaşık 1) ve kenarları lehçe ve sarkık kadar alev geçerek küçük parçalara cam lamelleri ayrı kırarak kavisli lamel parçaları olun. </li><li> X döller laevis yumurta in vitro veya doğal çiftleşme ve aşamaları gastrula kültürün içinden 18 - önceden aşamaya göre sıralama için 0.1x MBS (10 oda sıcaklığında 11 saat sonrası fertilizasyon); 10 embriyoların - orta gastrula (11.5 aşama 11) toplamak. </li><li> 3 / 4x NAM yaklaşık yarısı dolu bir petri embriyolar transfer. Ince forseps iki çift kullanarak, gastrulae gelen fertilizasyon (vitellin) membran kaldırmak. </li><li> Aktarım kil astarlı yemekleri 3 / 4x NAM (yaklaşık 2 ml) oocytes ve oluklar yukarıda açıklandığı gibi bireysel embriyolar karşılamak için gösterim üreten, kil bireysel izlenimler hareketsiz. </li><li> Kil astarlı yemekleri embriyolar transfer ve g kapalı hayvan kesim kapaklarıastrulae ince forseps iki çift kullanarak. Değil, sadece ekvator doku 18 hayvan kap ektoderm izole etmek için özen gösterin. </li><li> Oosit ile temas hayvan kapağın iç yüzeyi ile her bir oosite hayvan yarımkürede bir hayvan kapağını yerleştirin. 8 saat veya daha uzun - 6 oosit yüzeyine maruz iç tabaka ile açık kalabilir (lamel kontaklar olarak hayvan kapakları kil ektoderm düzleşme, bombeli cam lamel fragmanı uygulanması ve cama aşağı doğru basınç uygulayarak rekombinantlarını arada tutmak ). Alternatif olarak, yukarı doğru bakan iç yüzeyi ile bir kil girinti hayvansal takarak ve kapağı üzerinde bir oosit yerleştirmek; Not: kil, küçük uzantıları ile rekombinant sabitleyin. </li><li> 20 ° C&#39;de Kültür kontrol embriyolar tahlil için sahne istenen ulaştıkları kadar. </li><li> Lamel / kil kaldırılması ve forseps ve saç döngü ile ektoderm izole ederek ayrı rekombinant. </li><li> MEMFA 1 saat (% 3.8 için ektodermal fragmanları FixMEM [0,1 M MOPS pH 7.4, 2 mM EGTA ve 1 mM MgSO 4] formaldehit). </li><li> -20 ˚C etanol ve mağaza MEMFA transfer fragmanları (ve kontrol embriyolar). </li></ol> In situ Hibridizasyon tarafından Ektoderm içinde Yanıtın 4. Analizi (ISH) <ol><li> ISH için çözümler hazırlayın. <ol><li> 1x PBS hazırlayın: 0.01 M fosfat tamponlu tuz, NaCl 0.138 M, pH 7.4 </li><li> Hazırlama PBS-Tween (PTW): 1x PBS,% 0.1 Tween-20 </li><li> 100x Denhart solüsyonu hazırlayın: 2% BSA,% 2 polivinilpirolidon,% 2 Ficoll </li><li> Hibridizasyon tamponu hazırlayın:% 50 formamid, 5x SSC, 1 mg / ml maya RNA Torula, 1 ug / ml heparin, 1x Denhart solüsyonu,% 0.1 Tween-20,% 0.1 CHAPS, 10 mM EDTA, DEPC H2O </li><li> Hazırlama Maleik asit tampon (MAB): 100 mM maleik asit, 150 mM NaCI, pH 7.5 </li><li> (Çözünmesi 60 ˚C ısı) MAB,% 2 reaktifi engelleme: MAB + blok hazırlayın </li><li> 100 mM Tris: Alkalen Fosfataz (AP) Tampon hazırlayınpH 9.5, 50 mM MgCI2, 100 mM NaCI,% 0.1 Tween, dH 2 O </li></ol></li><li> RNA Probe hazırlayın. <ol><li> 1.5 ml tüp (50 ul reaksiyon) eklemek ticari bir RNA polimeraz kit kullanılarak ve karışımı-NTP dig: 25.5 ul DEPC, H2O, 10 ul 5X Transkripsiyon tamponu, 2.5 ul 10x dig-NTP karışımı, 5 ul 100 mM DTT, 2 ul RNAsin, 2 ul doğrusallaştırılmış DNA şablonu (~ 1 ug / ul), 3 ul RNA Polimeraz ve 90 dakika için 37 ° inkübe edin. </li><li> 2 ul RNA Polymerase ekleyin ve 60 dakika için 37 ° inkübe edin. </li><li> % 1 agaroz jeli üzerinde reaksiyon 2 ul kontrol edin. </li><li> 1 ul RQ1 RNase-barındırmayan DNase ilave edin ve 20 dakika boyunca 37 ° inkübe edin. </li><li> 50 ul DEPC H2O, 25 ul 10 M amonyum asetat ve 313 ul etanolün eklenmesi ile prob Çökelme. -20 ° C&#39;de saklayın, gece boyunca (O / N), daha sonra 20 dakika boyunca 13.800 x g&#39;de santrifüjleme ile geri RNA. Kısaca spin 500 ul% 75 etanol ile yıkayın, kaldırmaketanol ve kuru hava pelet izin verir. Ekle 50 ul DEPC H 2 O. </li><li> 0.5 ug / ul ~ arasında nihai bir konsantrasyona kadar Hibridizasyon Tamponu ekleyin. </li></ol></li><li> Hibridizasyon için Tissue hazırlayın. Aksi belirtilmediği sürece, her bir çözüm değişikliği tarif edildiği gibi (yaklaşık 4 mi) ile üst şişeler doldurulur. <ol><li> Yatay rocker, 10 dakika her biri için% 75 etanol / PTW, daha sonra% 50 etanol / PTW Şişelerden ve transfer embriyolar etanol çıkarın. </li><li> 5 dakika rocker üzerinde her biri için PTW üç kez yıkayın. </li><li> 10 ug PTW içinde / ul Proteinaz K tedavisi transfer; Kaya tüpleri dik 15 dk. </li><li> Dikey kaya tüpleri - 0.1 M Triethanolamine pH 7.8 iki kez 10 dakika her durulayın. </li><li> Tüplere 12.5 ul asetik anhidrit eklenir ve dikey olarak 5 dakika sallayın. 5 dakika boyunca ilave 12,5 ul asetik anhidrit ile tekrarlayın. </li><li> Rocker dk dikey PTW 5 yıkayın. </li><li> Rocker% 4 paraformaldehid 20 dakika içinde Refix. Isı Hibridizasyon Tampon60 ˚C ye. </li><li> 5 dakika rocker üzerinde her biri için PTW üç kez yıkayın. </li><li> 250 ul Hyb Buffer her tüpten PTW tüm ama ~ 1 ml çıkarın ve ekleyin; hafifçe karıştırın tüpler girdap. Kaya tüpleri dik 5 dk. </li><li> 60 ˚C Hyb Tamponu (0.5 mi) ile değiştirin ve 60 ° C&#39;de 10 dakika yavaşça çalkalayın. Taze Hyb Tampon ile değiştirin ve 60 ˚C iki 4 saat sonra çalkalayın. </li><li> Isı sondası 60 ° C (3 dakika) (0.5 ug Hyb Tamponu içinde / ml 1 ml). Hyb Buffer çıkarın ve tüpler probu ekleyin. O / N 60 ° C&#39;de yavaşça çalkalayın. </li></ol></li><li> Antikor için Tissue hazırlayın. <ol><li> 60 ° C&#39;ye ısınmaya Hyb Tampon ve 2 x SSC +% 0.1 Tween-20 çözümler. </li><li> (- 3x yeniden kullanım için 2 -20 ˚C de probları kaydetmek) Hyb Tampon ile prob çözümü değiştirin. 10 dakika boyunca 60 ° C&#39;de yıkanır. </li><li> 2x SSC-Tween 60 ° (20 dakika çalkalanarak her biri) üç kez yıkayın. </li><li> 0.2X SSC-Tween 60 ° (20 dakika çalkalanarak her biri) üç kez yıkayın. </li><li> Rocker üzerinde 15 dakika her yatay için MAB (RT) iki kez yıkayın. </li><li> 1 ml MAB + bloğu ekleyin. Rocker dikey 2 saat yıkayın. </li><li> 1 / 2.000 Anti-dioksijenin-AP içeren 1 ml MAB + blok transfer. 4 CO / N dikey sallayın. </li></ol></li><li> Renk Reaktif için Tissue hazırlayın. <ol><li> MAB ile antikor çözüm değiştirin; MAB 5 dakika her yatay rocker üç kez yıkayın. </li><li> MAB 1 saat her yatay rocker üç kez yıkayın. </li><li> AP Tampon 10 dakika içinde her yatay rocker iki kez yıkayın. </li><li> Birden-iyi plastik tepsi doku aktarın AP Buffer kaldırmak ve BM Mor reaktifmi (~ 1 ml). Reaksiyon O / N karanlık 5 dakika içinde ilerlemeye izin verin. </li><li> Uygun boyama elde edildiğinde, PTW ve şişelere doku aktarın. Rocker üzerinde PTW iki kez 10 dakika her yıkayın. </li><li> Rocker 4 CO / N at Bouin çözeltide sabitleyin. </li><li> RT&#39;de Bouin üç% 70 etanol /% 30 PTW yıkar ile yıkayın. T DevamGerekli 18 ise epi-aydınlatma ve bir mikroskop monte kamera, ağartma kullanarak o görüntü yakalama. </li></ol></li></ol> 5. Sib Seçimi ve Klonlama <ol><li> En yüksek aktivite ile seçin havuzu (ektodermal dokusunda büyük tepki). </li><li> (Yukarıda protokol bölümü 2 kullanılarak) önceki adımda elde edilen koloniler, en yüksek etkinliği olan havuzu için gliserol stokları (uygun yoğunluğa LB ile seyreltilmiş) ile yaklaşık onda biri ile on yeni plakaları plaka titre edilir. </li><li> Etkinlik tek koloni takip dek havuzu boyutunu azaltın. </li><li> Vektör standart primerler kullanılarak DNA sekansı elde edilir. </li></ol>

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<li>Lustig, K., Kroll, K., Sun, E., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+cloning+of+a+Xenopus+T.-related+gene+%28Xombi.%29+involved+in+mesodermal+patterning+and+blastopore+lip+formation%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Expression cloning of a Xenopus T.-related gene (Xombi.) involved in mesodermal patterning and blastopore lip formation.</a> Development. 122, 4001-4012 (1996).</li>
<li>Hsu, D., Economides, A., Wang, X., Eimon, P., Harland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Xenopus.+dorsalizing+factor+Gremlin+identifies+a+novel+family+of+secreted+proteins+that+antagonize+BMP+activities%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">The Xenopus. dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities.</a> Molecular Cell. 1 (5), 673-683 (1998).</li>
<li>Lustig, K., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+an+oocyte+expression+assay+to+reconstitute+inductive+signaling%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">Use of an oocyte expression assay to reconstitute inductive signaling.</a> Proc. Natl. Acad. Sci. USA. 92, 6234-6238 (1995).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Basic+Methods+in+Cellular+and+Molecular+Biology.+Molecular+Cloning%5BTitle%5D)+AND+%22JoVE+Science+Education+Database%22%5BJournal%5D)">Basic Methods in Cellular and Molecular Biology. Molecular Cloning.</a> JoVE Science Education Database. , (2015).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Basic+Methods+in+Cellular+and+Molecular+Biology.+Restriction+Enzyme+Digests%5BTitle%5D)+AND+%22JoVE+Science+Education+Database%22%5BJournal%5D)">Basic Methods in Cellular and Molecular Biology. Restriction Enzyme Digests.</a> JoVE Science Education Database. , (2015).</li>
<li>Viczian, A., Zuber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+Determination+Using+the+Animal+Cap+Transplant+%28ACT%29+Assay+in+Xenopus+laevis%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis.</a> J. Vis. Exp. (39), e1932(2010).</li>
<li>Sive, H., Grainger, R., Harland, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSive%2C+H+%22Early+development+of.Xenopus+laevis.%3A+A+laboratory+manual%22">Early development of.Xenopus laevis.: A laboratory manual</a>. , Cold Spring Harbor Laboratory Press Cold Spring Harbor. New York. (2000).</li>
<li>Zygar, C., Cook, T., Grainger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+activation+during+early+stages+of+lens+induction+in+Xenopus%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Gene activation during early stages of lens induction in Xenopus.</a> Development. 125, 3509-3519 (1998).</li>
<li>Medina-Martinez, O., Jamrich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Foxe+view+of+lens+development+and+disease%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Foxe view of lens development and disease.</a> Development. 134, 1455-1463 (2007).</li>
<li>Amin, N., Tandon, P., Osborne, N. E., Conlon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA-seq+in+the+tetraploid+Xenopus.laevis.+enables+genome-wide+insight+in+a+classic+developmental+biology+model+organism%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">RNA-seq in the tetraploid Xenopus.laevis. enables genome-wide insight in a classic developmental biology model organism.</a> Methods. 66, 398-409 (2014).</li>
<li>Gilchrist, M., Zorn, A., Voigt, J., Smith, J., Papalopulu, N., Amaya, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+a+large+set+of+full-length+clones+from+a+Xenopus+tropicalis+EST+project%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Defining a large set of full-length clones from a Xenopus tropicalis EST project.</a> Developmental Biology. 271, 498-516 (2004).</li>
<li>Karpinka, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenbase%2C+the+Xenopus.+model+organism+database%3B+new+virtualized+system%2C+data+types+and+genomes%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Xenbase, the Xenopus. model organism database; new virtualized system, data types and genomes.</a> Nucleic Acids Research. 43, D756-D763 (2015).</li>
<li>Zhang, S., Li, J., Lea, R., Amaya, E., Dorey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Functional+Genome-Wide+In+Vivo+Screen+Identifies+New+Regulators+of+Signaling+Pathways+during+Early+Xenopus+Embryogenesis%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">A Functional Genome-Wide In Vivo Screen Identifies New Regulators of Signaling Pathways during Early Xenopus Embryogenesis.</a> PLoS ONE. , e79469(2013).</li>
</ol>

The authors declare that they have no competing financial interests.

Yetkili ektoderm bir yanıt indükleme yeteneğine sahip genlerin fonksiyonel klonlanması için, burada açıklanan yöntem, gen ürünlerinin geniş tanımlamak için kullanılabilir. Bu yöntem ifade klonlama teknikleri ile doku uyaran deneyleri birleştirerek geçmiş iş üzerine genişletir. Bu RNA, enjeksiyonu takiben, doğrudan veya dolaylı olarak, faktör uyarıcı üretim kaynağı olarak, Xenopus oosit metabolik yolları kullanmaktadır. Bu ilgi, bir cDNA kütüphanesi veya klonları diğer toplama ...

Forward genetic approaches to identify genes of interest through their function or loss-of-function1,2 are an integral part of understanding complex patterning events in development. Coupled with powerful reverse genetic techniques available to an ever-widening array of systems and researchers3-5, it is now possible to identify genes with a key functional role in a pathway and then elucidate that function at the cellular level and in interaction with other gene products. One approach to functionally identifying genes of interest that has yielded many key findings in the past is expression cloning6,7.
Our recent aim8 was to identify early lens-inductive factors, since it has been demonstrated that initial steps in the vertebrate lens-inductive process occur as early as gastrula stages. To that end, we used the transiently lens-competent9 animal cap ectoderm (stage 11-11.510) of Xenopus embryos as responding tissue for induction, and the stage VI Xenopus oocyte as a source of production for the inducing factors.
The following protocol builds on the expression cloning and sib selection protocols of Smith and Harland6,7, also successfully used by others11-13. In our oocyte expression system (first utilized for production of inducing factors by Lustig and Kirschner14), pools of injected transcripts capable of directly or indirectly causing the oocytes to produce factors that elicit a lens-inductive response in animal cap ectoderm are selected for and identified. Since the system is useful for expressing secreted inducing molecules directly (oocyte-injected INHBB mRNA causes mesoderm induction in mesoderm-competent animal cap ectoderm8), we originally expected the screening procedure to be useful chiefly for identification of paracrine factors. However, since we identified a nuclear factor in our screen (ldb18), it is clear that the system can be used to identify a wide variety of molecules such as transcriptional or translational regulatory factors, miRNAs, cofactors, or juxtacrine factors.

<table><tbody><tr><td>12/101 Antibody</td><td>Developmental Studies Hybridoma Bank</td><td>12/101</td><td>Monoclonal antibody for detection of muscle tissue</td></tr><tr><td>20x SSC Buffer</td><td>Sigma</td><td>S6639</td><td>for ISH</td></tr><tr><td>Acetic anhydride</td><td>Sigma</td><td>A6404</td><td>for ISH</td></tr><tr><td>Anti-Dig-AP</td><td>Roche</td><td>11093274910</td><td>for ISH</td></tr><tr><td>Aurum Plasmid Mini Kit</td><td>Bio-Rad</td><td>732-6400</td><td>Plasmid DNA purification</td></tr><tr><td>Blocking Reagent</td><td>Roche</td><td>11096176001</td><td>for ISH</td></tr><tr><td>BM Purple</td><td>Roche</td><td>11442074001</td><td>for ISH</td></tr><tr><td>Boekel Hybridization Oven</td><td>Fisher Scientific</td><td>13-245-121</td><td>for ISH</td></tr><tr><td>Bouin&#039;s Solution</td><td>Sigma</td><td>HT10132</td><td>for ISH</td></tr><tr><td>BSA</td><td>Sigma</td><td>A9647</td><td>for OCM</td></tr><tr><td>CHAPS</td><td>Sigma</td><td>C3023</td><td>for ISH</td></tr><tr><td>Collagenase A</td><td>Roche</td><td>10103578001</td><td>Defolliculation of oocytes</td></tr><tr><td>Cysteine</td><td>Sigma</td><td>C121800</td><td>Dejelly embryos</td></tr><tr><td>DEPC-H2O</td><td>Fisher Scientific</td><td>BP5611</td><td>for ISH</td></tr><tr><td>Dig-RNA Labeling Mix</td><td>Roche</td><td>11277073910</td><td>for ISH probes</td></tr><tr><td>Dumont #5 forceps</td><td>World Precision Instruments</td><td>500233</td><td>for Vitelline envelope removal</td></tr><tr><td>Ethyl 3-aminobenzoate</td><td>Sigma</td><td>A5040</td><td>MS222 anesthetic</td></tr><tr><td>Ficoll PM 400</td><td>Sigma</td><td>F4375</td><td>for Injection media</td></tr><tr><td>Formamide</td><td>Sigma</td><td>F9037</td><td>for ISH</td></tr><tr><td>Gentamicin sulfate</td><td>Sigma</td><td>G1914</td><td>for OCM</td></tr><tr><td>Glass capillaries</td><td>World Precision Instruments</td><td>4878</td><td>3.5&quot; long, I,D, 0.530 mm</td></tr><tr><td>Glass sample vials</td><td>Fisher Scientific</td><td>06-408B</td><td>for ISH</td></tr><tr><td>Hair loop</td><td></td><td></td><td>Hair affixed in pasteur pipette for tissue manipulation</td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H4784</td><td>for ISH</td></tr><tr><td>Injector Nanoliter 2010</td><td>World Precision Instruments</td><td>Nanoliter 2010</td><td>Microprocessor-controlled microinjector</td></tr><tr><td>Instant Ocean</td><td>Carolina</td><td>972433</td><td>Aquarium Salt for frog recovery</td></tr><tr><td>IRBG XGC Xenopus verified full-length cam cDNA</td><td>Source Bioscience</td><td>989_IRBG</td><td>cDNA library&amp;nbsp;</td></tr><tr><td>LB Agar plates with 100 &amp;micro;g/ml Ampicillin</td><td>Teknova</td><td>L5004</td><td>150 mm pre-poured LB-Amp plates for sib selection</td></tr><tr><td>LB Luria Broth</td><td>Teknova</td><td>L8650</td><td>LB for collecting colonies in sib selection from plates and dilution of cultures</td></tr><tr><td>Magnetic mRNA Isolation Kit</td><td>New England BioLabs</td><td>S1550S</td><td>for isolation of poly(A)-enriched RNA</td></tr><tr><td>Maleic Acid</td><td>Sigma</td><td>M0375</td><td>for ISH</td></tr><tr><td>Manual Microfil Micromanipulator</td><td>World Precision Instruments</td><td>M3310R</td><td>Manual micromanipulator</td></tr><tr><td>Nutating Mixer</td><td>Fisher Scientific</td><td>22-363-152</td><td>Rocker for ISH</td></tr><tr><td>Permoplast</td><td>Nasco</td><td>SB33495M</td><td>Clay for injection and dissection dishes</td></tr><tr><td>Phosphate Buffered Saline</td><td>Sigma</td><td>P5368</td><td>for ISH</td></tr><tr><td>PMSG</td><td>Sigma</td><td>G4877</td><td>to stimulate oocyte development</td></tr><tr><td>Polyvinylpyrrolidone</td><td>Sigma</td><td>PVP40</td><td>for ISH</td></tr><tr><td>Programmable Puller</td><td>World Precision Instruments</td><td>PUL-1000</td><td>Micropipette needle puller</td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P6556</td><td>for ISH</td></tr><tr><td>pTnT Vector</td><td>Promega</td><td>L5610</td><td>cDNA library construction</td></tr><tr><td>Riboprobe Combination System</td><td>Promega</td><td>P1450</td><td>in vitro transcription</td></tr><tr><td>Superscript Full Length cDNA Library Construction Kit</td><td>Life Technologies</td><td>18248013</td><td>kit for cDNA library construction</td></tr><tr><td>Sutures, 3-0 silk</td><td>Fisher Scientific</td><td>19-037-516</td><td>Suture thread and needle for post-oocyte removal</td></tr><tr><td>Torula RNA</td><td>Sigma</td><td>R3629</td><td>for ISH</td></tr><tr><td>Triethanolamine</td><td>Sigma</td><td>T1502</td><td>for ISH</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td>for ISH</td></tr><tr><td>Universal RiboClone cDNA Synthesis System</td><td>Promega</td><td>C4360</td><td>alternative kit for cDNA library construction</td></tr><tr><td>Xenopus Full ORF Entry Clones - ORFeome Collaboration</td><td>Source Bioscience</td><td>5055_XenORFeome</td><td>ORFeome Clones</td></tr><tr><td>XL2-Blue Ultracompetent Cells</td><td>Agilent Technologies</td><td>200150</td><td>cells for transformation of cDNA library</td></tr></tbody></table>

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.

Hayvan kap doku yanıt, oositler enjekte mRNA ekspresyonu yanıt olarak in situ hibridizasyon (Şekil 2 ve Tablo 1) e göre otx2 ekspresyonu için tahlil edilmiştir; otx2 göz taslakları nöral tüp kapanması ile ilgili olası lens ektoderm (PLE) cinsinden ifade edilir kalınlaşma 19. Otx2 da PLE dışındaki ön nöral ektoderm olarak nöral olmayan baş ektoderm ifade edilir Bununla birlikte, bu nöra...

Watch this Scientific Journal Video about Functional Cloning Using a Xenopus Oocyte Expression System at JoVE.com

Functional Cloning Using a Xenopus Oocyte Expression System

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

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.

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

functional-cloning-using-a-xenopus-oocyte-expression-system

Research

JoVE Journal

Developmental Biology

Functional Cloning Using a Xenopus Oocyte Expression System

8.0K Görüntüleme.  Shepherd University. 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.

Video: Functional Cloning Using a Xenopus Oocyte Expression System

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.

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 ...

Gelişim Biyolojisi

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 ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

CRISPR/Cas9-tabanlı Xenopus Leapfrogging için ilkel Germ hücre transplantasyonu

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

Genetik

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, ...

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

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 ...

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 ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

Sperm Yerine Fare Partenogenetik Haploid Embriyonik Kök Hücrelerin Uygulanması

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

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