This research was supported by the Australian Research Council grant CEO348212 and the University of Newcastle. The assistance of Dr. Sam Zhang is acknowledged.

1. Zigotik Embriyo Gelişimi <ol><li> Bitki Materyali <ol><li> Büyüme Medicago, bir 14 saatlik aydınlık periyoduyla serada 23 ° C / 19 ° C gündüz / gece sıcaklığı yabani tip Jemalong ya da yüksek oranda yeniden generable onun yakınındaki izogenik, (2HA olarak da bilinir) genotip Jemalong 2HA 13 truncatula. <ol><li> Suyun tohumu girin ve bir gece suda bekletin izin, böylece tohumun ekim önce Pierce (23 G iğne ile), tohum kaplama yüzeyi. Tam tohum örtecek kadar su ekleyin. </li><li> Saksı karışımı içinde her 15 cm çapında pota 3 tohum ekmek (10 kapların toplam) (kaba kum, perlit, lif-turba (1: 1: 1) artı Osmocote Exact Standardı 5 g: Yavaş salimli gübre) ve aktarma sera. Bitki tırmanmak sapları için pot ve çevre başına 1 bitki bir kafes tarafından var bu yüzden çimlenme sonrası sağlıklı fide saklayın. </li></ol></li></ol></li><li> Hasat Bölmeler <ol><li> W gelen bakla toplayınJemalong veya 2HA uygun erken embriyo aşamalarında elde etmek ild türü olarak ilk 9 nitelendirdi. Not: Farklı bölme aşamaları, Şekil 1 &#39;de gösterilmiştir ve karşılık gelen embriyo aşamaları, Şekil 2&#39;de gösterilmiştir. </li><li> Ayrı aşamaları 6 ve 7 (Tablo 2) yardım etmek için sahneye 6 geçen süreyi. Tablo 1 gereğince kriterleri kullanılarak hasat farklı pod aşamalarını kontrol ölçün. </li></ol></li><li> Bölmelerinden ovüllerin Kesme ve embriyo aşaması kontrol <ol><li> Gerekirse tek başına ince forseps kullanarak bakla veya stereo mikroskop ve forseps gelen ovüllerin izole edin. Gerekirse embriyo aşaması hızla standart bileşik mikroskobu (Şekil 3B) kullanılarak kontrol edilebilir. </li><li> Arabic 7.5 g sakız, 100 g klor hidrat, 5 ml gliserol, damıtılmış deiyonize su 60 ml su ihtiva eden Hoyer çözeltisi modifiye hazırlayın. 3-5 saat veya gece boyunca karıştırılarak çözülür. </li><li> Daha fazla rafine içind mikroskopi modifiye Hoyer çözümü 15,16 disseke ovüllerin temizleyin. Dikkatle berrak olana kadar oda sıcaklığında (ovül karşılamak için yeterli) Hoyer çözeltisi, küçük bir hacim içinde sağlam ovüllerin ve yeri al. </li><li> Diferansiyel girişim kontrast (DIC) optik ile örnekleri görüntüleyin. Bir dijital fotoğraf makinesi 9 (Şekil 2) iwth görüntüler çekin. </li><li> Pod merkezi bölgesinde en üniforma ovüllerin edinin ve gerekli sayıda birikir. Bir örnek, Şekil 3A&#39;da gösterilmiştir. Daha fazla analiz için gerekli sıcaklıkta buz ve mağaza ovüllerin (-80 ° C için nükleik asitler) toplayın. NOT: histolojik kesitlerin analizi, transmisyon elektron mikroskobu ile ilgili detaylar için, ve in situ melezleme kağıtları 8,9 bakınız. </li></ol></li></ol> İn Vitro 2. Somatik Embriyo Gelişimi <ol><li> Bir M. kullanın truncatula çeşidi tŞapka kolaylıkla kültür embriyoları meydana edebilmektedir. Yaprak eksplantlarından için bu protokol için 2-4 aylık olduklarında 2HA bitkiler 13,17 kullanın. </li><li> Bir uzatma kök son neredeyse tamamen genişletilmiş üç yapraklı yapraktan broşürler alın. </li><li> Dehidratasyonu önlemek için bir kültür tencerede nemli kağıt havlu üzerinde üç yapraklı yaprakları tutun. NOT: üç yapraklı yaprak hasat edilir sonra da en az gecikmeyle kullanılabilir olması gerekir. </li><li> Kültür ortamının hazırlanması <ol><li> 4: 1: agar 5 orta (ağırlık% 0,8: hacim) içinde 9 cm Petri kapları P4 10 kullanın. Not: P4 10: 4: 1: 5, orta P4 bazal ortamı 18 artı 10 uM 1-naftalenasetik asit (NAA), 4 uM 6-benzilaminopurin (BAP), 1 uM absisik asit (ABA) ve 5 uM gibberellik oluşmaktadır asit (GA). GA + ABA kullanımı embriyo oluşumunu hızlandırır ve embriyo sayısı 14 maddi artışlara neden olmaktadır. </li><li> 1 L P4 bazal ortamı Makyaj; (calciu yaptığı büyük tuzlarından oluşan,) ayrı ayrı m minör tuzları ve vitaminler (Tablo 3), şelatlanmış demir (ayrı makyaj), kazamino asitler (kasein hidrolizat), 30 g sakroz, 8 g agar. </li><li> Uygun parçalar halinde -20 ° C&#39;de ana tuzlar, kalsiyum, kazamino asitler, minör tuzları ve vitaminler mağazası stok çözeltileri. 4 ° C&#39;de şelatlı demir saklayın. </li><li> • 7H FeSO 4 1,853 g eklenirken karıştırılırken iyonu giderilmiş damıtma suyu yaklaşık 900 ml Na2EDTA • 7.44 g 2H 2 O çözündürülmesiyle şelatlı demir (200x) makyaj, daha sonra 98-99 ° C&#39;ye kadar bir çözüm getirmek 2 O. Terkip maddeleri, çözündüğü zaman son olarak 1 L tamamlanır. Amber içinde saklayın 4 ° C&#39;de şişe renkli. </li><li> Tablo 4&#39;de olduğu gibi hisse senedi çözümleri ile P4 bazal ortamı Makyaj. Ortamda hormonlar 10 uM NAA, 4 iM BAP, 1 uM ABA, 5 mcM GA (Tablo 4 ve Tablo Spesifik Malzeme bakınız). NAA Ekleve önceki BAP otoklavda bir şınngaya bağlanmış bir 0.22 um filtre ile filtre sterilizasyonu, otoklav yaklaşık 55 ° C&#39;ye kadar soğutulduktan sonra, ABA ve GA ekleyin. Nazik dönen ile iyice karıştırın. </li><li> Otoklav öncesinde 1 M KOH ile pH 5.8 medyayı ayarlayın. 20 dakika boyunca 121 ° C&#39;de otoklav. </li><li> Filtre sterilize ABA ve GA, ekleme ve bir laminer akış kaputu ya da biyolojik tehlike kabine steril 9 cm Petri kapları içine medya yaklaşık 25 ml dökün otoklav sonra. Serin ve ağar kapaklı kapalı olarak ayarlamanıza olanak tanır. Sonra kapağı üzerine koymak ve kapağın üzerinde hiçbir yoğuşma olmadığından emin olun. (Kapak kondens önlemek için bir karton kutu içinde) 4 ° C&#39;de gerekli ve mağaza gibi etiket. </li></ol></li><li> Yaprak dokusu sterilizasyonu <ol><li> UV-sterilize laminer akış kaputu ya da biyolojik tehlike kabine çalışın. </li><li> Yeri önceden otoklavlanmış yay çay demlik mesh topun içine bırakır. </li><li> Bir cul etanol:% 70 (v v) yaprakları içeren çay demliği daldırın30 saniye boyunca Ture pot. NOT: Bu prosedürde tüm adımları kullanın 250 ml otoklavlanırlar kapak polikarbonat kültür tencere vida. </li><li> Boşaltın ve daha sonra transferi ve 1 yaprak dokusu sokmak: 8 (hac: hac) ile seyreltilmiş ağartma maddesi (% 0.5 klorin), 10 dakika boyunca karıştırılır. Zaman zaman hafifçe karıştırılır. </li><li> Çamaşır suyu boşaltın ve steril deiyonize distile su içeren bir kültür tencerede çay demliği aktarın. Yavaşça girdap ve fazla suyu boşaltın. Steril deiyonize distile su taze kültür pot ile bir kez daha tekrarlayın. </li><li> Steril deiyonize damıtılmış su, taze kültür kap steril forseps çay infüzyon edicinin yaprakları çıkarın. Steril kapağını tekrar ve tersini ve dönen tarafından durulayın. Eksplantlar hazırlamak için hazır olana kadar steril su üzerinde yüzen yapraklar bırakın. </li></ol></li><li> Eksplantlarındaki hazırlanması ve Kaplama <ol><li> Steril teknikleri kullanarak, bir neşter ile kenarından bireysel sterilize broşürler Döşeme ve iki ya da thr içine kalan dikdörtgen kesilmişdikdörtgen eksplantlar her eksplant (Şekil 5A) merkezinde orta damar ile (8-10 x 3-5 mm) ee. NOT: eksplant büyüklüğü ilk Kalluslaştırma başlatılması oldukça önemlidir. Take-away gıda kapları steril kapaklarındaki kesme yürütmek. </li><li> Plaka 9 cm çapında Petri kapları (Şekil 5B) agar-katılaşmış kültür ortamında 6 eksplantlar. Her zamanki yaprak yönünü korumak için aşağı eksplantlar abaxial tarafı Plate. </li><li> Yemeğin etrafında gergin Parafilm (Şekil 5C) ile Petri kapları mühür. Doku şimdi inkübasyon için hazırdır. </li></ol></li><li> Doku Kuluçka <ol><li> Kültür süresi boyunca sıcaklığı kontrol edilen bir oda ya da büyüme odasında 28 ° C&#39;de karanlıkta inkübe edin. NOT: eksplantlar, dediferansiyonunu başlatmak hücre bölünmesi, çoğalması (kallus oluşumu) ve embriyogenez (farklılaşma) uğrayacaktır. </li><li> Farklılaştırarak expla altkültürütaze ortama 3 hafta sonra NTS. Bu altkültür bir laminer akış kaputu ya da biyolojik tehlike kabinede, steril paslanmaz çelik spatula ve forseps kullanılarak tüm eksplant aktarın. Kalluslu oluşur ve kallus Şekil 5C büyük bir boyutunu aşıyor gibi taze ortama alt kültüre zaman bireysel yara dokusunun% 50 transfer. </li><li> Yaklaşık 4 hafta içinde ilk embriyoların uyun. Senkronize bir derece olsa da, 4-8 haftalık kuluçka dönemi boyunca farklı embriyo aşamalarını toplamak (Şekil 6A, B). Deneysel hedefleri doğrultusunda bir mikroskop ve işlem altında toplayın. </li><li> Eksplantlar 3 ay boyunca embriyo üretmeye devam edeceğiz, böylece her 3 haftada bir altkültürü ve periyodik embriyolar kaldırmak. </li></ol></li></ol>

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The authors declare that they have no competing interests.

açıklanan protokoller oldukça yalındır ve tüm çağdaş hücre ve moleküler teknikler ile baklagil embriyogenezde soruşturma izin verir. Bu avantaj ve in vivo ve in vitro yaklaşımlar hem de dezavantajları vardır tanır. Hem olgunlaşmamış tohumlar 19 kültürüne göre erken embriyogenez daha fazla odaklanmak sağlar. In vivo çalışmalarda durumunda neler açıklanan ağırlıklı birçok embriyo çalışmaları için uygundur b?...

Hasat alan ve toplam üretim 1&#39;de hububat ikinci baklagiller, yaklaşık 20.000 tür ve Leguminosae (Fabaceae veya) ailesi ile yüksek bitkilerin üçüncü büyük aileyiz vardır. Soya üçüncü büyük kültür bitkisidir. Tahıl baklagiller diyet proteini üçte biri ve insan tüketimine 2 bitkisel yağ üçte biri ile ilgili sağlar. Onların N 2 sabitleme kapasitesi ile Baklagiller ayrıca tohum kotiledonlarında soya, mağazalar protein ve yağ gibi. Sürdürülebilir tarımsal sistemlere Medicago truncatula katkıda önemli genetik ve genomik kaynakların 3,4 ile genetik ve genomik baklagil modelidir. M. iken truncatula giderek baklagil tohum biyolojisi 5-7 ve embriyojenezi 8,9 incelemek için istihdam edilmiştir baklagil-rhizobium simbiyoz 4 anlamada ilerlemeler sağladı. Arabidopsis embryogenesis yoğun 10,11 okudu ama ben olmuştursa olmayan baklagil ve embriyogenez ayrıntıları Medicago&#39;dan 8,10 özdeş değildir. M&#39;de zigotik embryogenesis truncatula farklı bir çok hücreli hipofiz, bir endoployploid uyutucuya ve bazal transferi hücresinin 8, ilginç özelliklere sahiptir. Somatik embriyojenez (SE), yaygın olarak bitkiler 12 rejenerasyonu için kullanılır. Baklagil modelinde M. In truncatula, ebeveyn Jemalong dan geliştirilmiştir tohum çizgisi Jemalong 2HA (2HA) somatik embriyogenez 13 yüksek oranlarına sahip. üretilen embriyoların sayısı son zamanlarda önemli ölçüde köklü orta 14 hem gibberelik asit (GA) ve absisik asit (ABA) eklenerek artış olmuştur. GA ve ABA genellikle antagonistik 14 hareket verilen sıradışı sinerjik bu durumda GA ve ABA hareket içinde. kallustan embriyoların embriyogenez evresi hali hazırda visuall saptanmasına olanak sağlamaktadır yüzeyi üzerinde gelişebiliry ve kolaylıkla hasat edildi. Somatik embriyojenez incelenmesi (Jemalong) (2HA) embriyonik ve embriyonik olmayan olan izojenik hatlarının yakınında kolaylaştırır olması ve in vivo ve farklı deneysel olanaklar sağlar in vitro sistemlerde de sahip. Embriyo gelişimi hücresel ve moleküler mekanizmaların anlaşılması anlayış tohum ve bitki gelişimi için gereklidir. Baklagiller, diğer çeneklilere olduğu gibi, insan beslenmesi için kullanılan ürünler depolamak embriyo kotiledonları olduğunu. Erken embryogenesis hızlı hücre bölünmesi ve doğru embriyo desenlendirme içerir. 8 gün sonra gübreleme, M&#39;de truncatula embriyo erken kotiledon aşamaları ulaşır. morfolojik karakterizasyon tam sera koşullarında döllenme sonrasında gün gösterilmez. Böylece, gelişmekte olan embriyo aşamasına belirtmek için verimli bir standart yaklaşım, genetik REGUL okuyan değerlidirErken zigotik embriyojenezin tirme. Bu yazıda, baklagil modelde M. de embriyogenezin biyolojik çalışmalar için gelişen embriyolar toplamak için iki standart protokolleri sağlamak truncatula. İlki ikinci kolayca erişilen büyük embriyo sayıları sağlamak için kültür yaprak eksplantlarından yoluyla somatik embriyogenesis ise embriyogenez ve pod morfolojisi ilişkilendirerek Zigotik embriyolar toplamaktır.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >P4 medium</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Use Sigma-Aldrich Chemicals or other analytical grade supplier</td>
		</tr>
		<tr >
			<td >Major salts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Minor salts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Vitamins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Agar</td>
			<td>Bacto Laboratories</td>
			<td>214010</td>
			<td>Bacto agar</td>
		</tr>
		<tr >
			<td >Plant hormones</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1-Naphthaleneacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>N0640</td>
			<td>Dissolve in small amount of 1 M NaOH</td>
		</tr>
		<tr >
			<td >Abscisic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A1049</td>
			<td>Dissolve in small amount of 1 M NaOH</td>
		</tr>
		<tr >
			<td >6-Benzylaminopurine</td>
			<td>Sigma-Aldrich</td>
			<td>B3274</td>
			<td>Dissolve in MQ water with heating and few drops 1N HCl</td>
		</tr>
		<tr >
			<td >Gibberellic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>G7645</td>
			<td>Dissolve in small amount of ethanol</td>
		</tr>
		<tr >
			<td >Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Stereo dissecting microscope</td>
			<td>Leica</td>
			<td>MZFLIII</td>
			<td>Or similar</td>
		</tr>
		<tr >
			<td >Light microscope</td>
			<td>Zeiss</td>
			<td>Axiophot</td>
			<td>Or similar, with suitable optics</td>
		</tr>
		<tr >
			<td >Digital camera</td>
			<td>Zeiss</td>
			<td>AxioCam HRc</td>
			<td>Or similar</td>
		</tr>
		<tr >
			<td >Sterilising leaves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >250 mL screw cap polycarbonate container with polypropylene lid</td>
			<td>SARSTEDT</td>
			<td>75.9922.519</td>
			<td>Autoclavable</td>
		</tr>
	</tbody>
</table>

protocols for obtaining zygotic and somatic embryos for studying the regulation of early embryo development in the model legume medicago truncatula

Tek bir hücre zigot başlayarak Erken embryogenesis hızlı hücre bölünmesi ve morfogenez geçer ve morfolojik öncesi küresel, küresel, kalp, torpido ve kotiledon aşamalarında ile karakterizedir. Bu ilerici gelişme karmaşık bir moleküler ağ sıkı yönetmelik altındadır. Benzer bir gelişme aşamasında yeterli bir erken embriyolar Hasat erken embriyogenez hücresel ve moleküler düzenleme incelemek için gereklidir. Medicago&#39;dan örneğin 8 gün erken kotiledon aşamaya ulaşmak için truncatula ise erken embriyogenez kısa hızlı morfolojilerinden uğrar çünkü bu kolay değildir. Burada, iki yaklaşımlarla sorunu gidermek. İlki zigotik embriyo aşamasına işaret yardımcı embriyo gelişimi ve bakla morfolojisi arasında bir bağlantı kurar. Bu, özellikle bakla spiraller ve dikenler geliştirilmesi sayısına dayanır. In vivo s tamamlayacak alternatif bir yoltudies somatik embriyoların üretilmesi için kültür yaprak eksplantlarından yoluyladır. Orta sıra dışı bir hormon kombinasyonu içeren - bir oksin (1-naftalenasetik asit), bir sitokinin (6-benzilaminopurin), absisik asit ve gibberellik asit. farklı aşamalarında diseksiyon olmadan kallus dışında büyüyen ayırt edilebilir.

F - Farklı embriyo aşamaları Şekil 2A gösterilmiştir ise F - Farklı embriyo aşamalarına karşılık gelen zigotik embryogenesis farklı pod yapılarda Şekil 1A gösterilmiştir. Aynı aşamada bakla seçerek, Yumurtacığa örnekleri oldukça tekdüze (Şekil 3A) elde edilebilir ki. RT-qPCR embriyoyu kullanarak belirli genler kolayca algılanır ve zaman derste 9 değerlendirilebilir. Bazı ek diseksiyon embriyo <st...

Watch this Scientific Journal Video about Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula at JoVE.co

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

Model Baklagil Erken Embriyo Geliştirme Yönetmeliği incelenmesi için zigotik ve somatik embriyolar Elde protokolleri<em&gt; Medicago truncatula</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

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Research

JoVE Journal

Developmental Biology

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

13.8K Görüntüleme.  University of Newcastle, Callaghan, New South Wales, Australia. The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided. 

Video: Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Evans Blue Dye ile Zebra balığı Larva İskelet Kası Bütünlüğü Analizi

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Gelişim Biyolojisi

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

Yaşayan Zebra balığı Embriyo Hücre alt yapıları görüntüleme

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Transcriptome Profil<em&gt; In-vivo</emİki renkli Mikroarray Platformu Kullanma&gt; Üretilen Sığır pre-implantasyon Embriyolar

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Zebra balığı erken embriyo ve tümör hücresel elektrik aktivitesinin görselleştirme

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

En düşük Invaziv embriyo transferi ve tavşan modelinde optimum embriyo aşamasında embriyo vitrifikasyon

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

Erken Fare Embriyolarından Sıralama için Küçük RNA Kütüphanelerinin Hazırlanması

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

Erken Drosophila Embriyosunda İmmünohistokimya ve RNA In-situ Dağılımı için Bir Protokol

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

Zebra Balığı Embriyoları İçin Basit ve Etkili Bir Transplantasyon Cihazı

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Sıçan Yağ Dokusu Mezenkimal Kök Hücrelerinin İnsülin Üreten Hücrelere Farklılaşması İçin İzolasyonu

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

En Yüz Fare Embriyolarında Düzlemsel Morfogenez Analizi için Endokardiyal Minderin Hazırlanması

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart ...