Yazarlar protokol gelişiminde ön çalışmaları için Gregory Haller (Mount Saint Vincent Üniversitesi) teşekkür etmek istiyorum. Yazarlar ayrıca onun teknik uzmanlık ve filme ve üretim el yazması ses/görüntü bölümü ile ilgili yardım için Nicholas Jones (Mount Saint Vincent Üniversitesi) teşekkür etmek istiyorum. Daniel Andrews MSVU ve Doğa Bilimleri ve Mühendislik Araştırma Konseyi, Kanada (NSERC) yolu ile bir lisans öğrenci Araştırma Ödülü fon tarafından desteklenmiştir. Tamara A. Franz-Odendaal bir NSERC bulma Grant tarafından desteklenir.

Not: embriyo aşamaları için tablo hazırlama Hamburger ve Hamilton12 (HH) kullanmaktadır.
1. embriyo kuluçka
<ol><li>Steril, ısı kontrollü kuluçka 37 ° C ± 1 ° C ve ~ %40 Nem, döllenmiş tavuk yumurtası kuluçkaya.</li>
	<li>Yumurta 1 çevirmek her gün ve HH34 için kuluçkaya kurmalarına izin (8 gün sonrası döllenme).</li>
</ol>2. hazırlık ve sterilizasyon malzemeleri
<ol><li>12 embriyolar, distile su, % 0,85 piliç salin, 12 Cam pipetler, 1 kutu kağıt mendil, yarı gözenekli filtre kağıdı karelerinin 24 2.5 cm x 2,5 cm ve 24 2.5 cm x 2,5 cm kare çelik tel 1 L, 2 L mesh aşağıya doğru kavisli kenarlar ile basınçlı kap için. Malzemeler, sterilize etmek otoklav 121 ° c en az 15 dakika onları. Not: Malzemelerin ısıyla önceden yapılabilir.</li>
	<li>Kuluçka makinesi ile % 70 etanol yan, raflar ve kapı silerek sterilize. Place 2 plastik kaplar autoclaved içeren distile su içinde nem korumak için kuluçka makinesi. 37 ° C ± 1 ° C ve ~ %40 nem için kültür kuluçka prewarm. Kuluçka makinesi tamamen karanlık olduğundan emin olun; Kapıyı şeffaf ise, folyo ile kapak.</li>
	<li>100 mL besin orta ve 1 mL penisilin kültür kuluçka makinesine koyarak prewarm.</li>
	<li>Bir kağıt havlu ile çalışma alanı kapsayacak ve % 70 etanol ile püskürtülerek tezgah sterilize. Forseps, diseksiyon makası, jilet ve % 70 etanol plastik bir kaşıkla 2 çift benzer bir şekilde sterilize.</li>
	<li>Yer 12 steril 100 mm Petri yemekler ve 24 35 mm Steril Petri yemekler kullanmak için bankta. Bulaşıkları bir steril torba kadar kullanmak kalır emin olun.</li>
</ol>3. embriyo hazırlık
Not: Bu noktadan itibaren kültürler kirletici önlemek için koruyucu toz yüzünü maske takmak. Bir bakteri veya mantar enfeksiyonu kültür berbat olacak ve bunu hızlı bir şekilde kuluçka makinesi bütün kültürlerde yayılan bir risk olduğunu.
<ol><li>Çatlamak yumurta açın ve embriyo %0.85 piliç serum fizyolojik içeren bir 100 mm Petri kabına aktarın. Anatomik özellikleri göre embriyo Hamburger ve yönergeleri12 basamak Hamilton'da açıklanan ve embriyo onaylamak HH34 aşamasıdır.</li>
	<li>Boyun kesme ve steril bir jilet kullanarak orta hat, embriyo Başkanı bisect. Steril forseps kullanarak, beyin kaldırın. Gaga olduğu gibi bırakma.</li>
</ol>4. kültür Kur
<ol><li>35 mm Petri yemekler bir 100 mm Petri kabına içinde yer 2.</li>
	<li>Çelik tel örgü içinde her 35 mm Petri kabına yerleştirin.</li>
	<li>Forseps kullanarak, 1 bisected başkanları 2.5 cm x 2,5 cm yarı gözenekli filtre kağıt parçası için yukarı dönük olacak şekilde göz aktarabilirsiniz. Doku güvenli ve kapalı hafifçe eğerek kayma değil emin olun.</li>
	<li>Forseps kullanarak, dikkatlice göz doku ve filtre kağıdı 1 35 mm Petri yemekler, üst göz dokusuyla yükseltilmiş bir sahne oluşturma çelik tel örgü üstüne koyun.</li>
	<li>4.3 ve diğer yarısı kafa ile 4.4 numaralı adımları yineleyin.</li>
	<li>Filtre kağıdı düzeyine ulaşıncaya kadar steril cam pipet kullanarak dikkatle her 35 mm Petri kabına doğrudan besin orta ekleyin. Besin orta yarım baş daldırın değil.</li>
	<li>10.000 U/mL penisilin-streptomisin 50 µL her Petri kabına besin ortamda ekleyin.</li>
	<li>Doku kağıt küçük bir kare kat ve 100 mm içinde Petri kabına yerleştirin. Autoclaved distile suyla nemlendirin.</li>
	<li>Kültür çanak 4 gün için 37 ° C ± 1 ° C ve ~ %40 Nem, steril, karanlık kuluçka yerleştirin.</li>
	<li>Her embriyo için 3 ve 4 numaralı adımları yineleyin. Not: Embriyo sayısı belirli denemeyi bağlı olacaktır; burada, biz 12 embriyolar için protokol tanımlamak.</li>
</ol>5. kültür bakım
<ol><li>Bir kez günlük, taze, autoclaved distile su ile kuluçka plastik kaplar kontör.</li>
	<li>Günlük tüm kültürler için bakteriyel veya mantar enfeksiyonları kontrol. Bu parçalanmış veya orta rengi değişti kültürler bulaşmış. Bulaşmış bütün kültürler atmak.</li>
</ol>6. fiksasyon
<ol><li>Kültür, aşağıdaki tüm yemekler kuluçka makinesi kaldırabilirsiniz.</li>
	<li>Forseps bir çift kullanarak, doku gözyaşı için dikkat çekici filtre kağıdı--dan göz yavaşça çıkarın.</li>
	<li>Göz doku %4 paraformaldehyde 1 fosfat tamponlu tuz gecede 4 ° C'de x veya % 10 formalin gecede, oda sıcaklığında tarafsız tamponlanmış düzeltin.</li>
	<li>Doku 4 ° C'de 1 fosfat tamponlu tuz x saklayın.</li>
</ol>

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Yazarlar ifşa gerek yok.

Bu iletişim kuralı bir tavuk gözünden embriyonik gün 8 (HH34) büyüme elde etmek için kurulan doku kültürü teknikleri kullanır vitro 4 gün için. Bu kılavuz kültür Yöntem aslında Trowell15tarafından tanımlanmıştır. Biz en iyi duruma getirilmiş bir protokol kullanan embriyonik tavuk göz15ayrı doku katmanları arasında endüktif sinyalleri çalışma için kılavuz ile yarı geçirgen bir zar olan Pinto ve Hall'ın 1991 çalışma<sup class=...

Embriyonik tavuk (Gallus gallus) biyoloji alanında yaygın olarak kullanılan bir mükemmel model organizmadır. Kuluçka süresinin yaklaşık 21 gün olduğunu ve birçok yumurta aynı anda, hızlı ve verimli deneme yapma inkübe. Belki de en önemlisi, embriyo da kolaylıkla, yoğun çalışma temel gelişimsel süreçleri ve genler ve proteinler bu işlemler sürücü etkinleştirme yönetilebilir.
Embriyonik tavuk göz üzerinden farklı dokuların birçok diğer vücut sistemleri için benzer bir dizi etkileşim gelişir karmaşık bir organdır. Bu yöntem, gelişmiş gelişim aşamasında özellikle bu dokuların gelişimi çalışma sağlar. Örneğin, çok katmanlı retina sinir sisteminin gelişimi eğitim görenler için özel ilgi olabilir. Kornea, vitreal vücut, objektif, sklera ve göz kapakları gibi diğer göz dokuların çalışma alternatif yöntemleri araştırmacılar yararı vardır. Tavuk embriyonik göz de düz kemikleri, Intramembranous kemik indüksiyon ve ossifikasyon omurgalılar1çalışma için bir model olarak kullanılan scleral kemikçiklerdeki bir dizi içerir.
Şu anda, embriyonik geliştirme eğitim için kullanılan bir yöntem vardır. İnhibitör antikorlar veya diğer inhibitör molekülleri2,3, microinjections cerrahi olarak inhibitörü4' te batırılmış lastikteki implante ve adım5 tümleyici genler için kullanılabilir tüm yöntem vardır ya da bir embriyo ilgi proteinler. Benzer yöntemleri upregulate proteinler için kullanılır. Bu yöntemleri sınırlamaları değildir. Örneğin, kimyasallar embriyonik gelişim alter için kullanıldığında, embriyo üzerinde ölümcül etkileri değerlendirilecek gerekir ve bu uygulamanın, Beka sağlamak için düşük dozda yerelleştirilmiş sitelere yukarıda belirtilen yöntemlerden kısıtlar embriyo.
Vitro doku kültürü organizmalar geniş bir geliştirme eğitim için kullanılan ve bazı yukarıda belirtilen sınırlamaları bypass için kullanılabilir. Örneğin, femora6, tüy tomurcukları7,8ve bacaklarda9 tavuk tüm doku kültürü yöntemleri, fare10 ve kökleri testis ve köklerini bitkiler11olarak kullanarak incelenmiştir. Bu yöntemler bilim adamları yüksek derecede sıcaklık dalgalanma ve besin durumu değiştirme yeteneği gibi doku geliştirme üzerinde kontrol vermek. Bütün embriyo dokudan yalıtım da böylece düzenleme çalışmaları daha yüksek konsantrasyonlarda küresel ölçekte etkinleştirme kimyasalların ölümcül etkileri çok daha az duyarlı kolaylaştırır. Vitro kültürü bir diğer önemli avantajı doku'nın hücresel ortamı korunması olduğunu; dokuların düzenleme farklı doku türlerine9arasındaki etkileşimler çalışma edinerek nispeten değişmeden kalır. Böylece, deneysel açılır kapılar için ek kültür vitro değil vivo içinde veya ovo içinde modelleri mevcuttur yaklaşıyor.
Şu anda, kimyasallar kullanarak embriyonik tavuk göz gelişimi eğitimi özellikle meydan okuyor. Bir dizi extraembryonic membranlar lastikteki veya kimyasal uygulamak üzere embriyo, kapak; o zaten zor bir yöntem daha da zorlaştıran, büyüdükçe embriyo Ayrıca yumurta içinde çok aktif. Bu iletişim kuralı göz ve aynı zamanda göz içinde gelişim süreçlerini incelemek için yeni fırsatlar sağlarken bu engelleri ortadan kaldırarak onun çevre dokular kolay erişim sağlar. Bu iletişim kuralı embriyonik göz içinde scleral kemikçiklerdeki indüksiyon çalışmaya kurulmuştur.

<table border="0" cellpadding="0" cellspacing="0" style="width:1081px;" width="1082">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm cell culture petri dishes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">353001</td>
			<td style="width:676px;">easy grip tissue culture dish, polystyrene, non-pyrogenic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 mm cell culture petri dishes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">353003</td>
			<td>tissue culture dish, polystyrene, non-pyrogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paper tissue</td>
			<td style="width:71px;">Kimtech</td>
			<td style="width:119px;">34155</td>
			<td>Kimtech Science Brand Task Wipers, 280 per carton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">wire mesh</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>stainless steel wire mesh (grid size 0.7 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">disposable glass pipettes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">14673-010</td>
			<td>Borosilicate glass disposable 5 3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">nutrient medium</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">12591-038</td>
			<td>Fitton-Jackson Modification, [+] L-glutamine with phenol red (BGJB)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">penicillin-streptomycin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4458</td>
			<td>10000 units/mL penicillin streptomycin solution stabilised</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">filter paper</td>
			<td style="width:71px;">Whatman</td>
			<td style="width:119px;">1454 090</td>
			<td>semi-porous filter paper 90mm</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">fertilized chicken eggs</td>
			<td style="width:71px;">Dalhousie University Agricultural College</td>
			<td style="width:119px;">n/a</td>
			<td>can be obtained from local farms</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium chloride (NaCl)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0420-3</td>
			<td>sodium chloride crystals, reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 L glass bottle</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">89000-240</td>
			<td>1 L pyrex autoclavable glass bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ethanol</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>BP82011</td>
			<td>70% molecular biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tupperware containers</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought and sterilized with EtOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">disposable razor blades</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">55411-050</td>
			<td>single edge industrial razor blades (surgical carbon steel)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic spoons</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought and sterilized with EtOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dust mask</td>
			<td style="width:71px;">3M</td>
			<td style="width:119px;">n/a</td>
			<td>3M 8500 Comfort Mask</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">paraformaldehyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td>paraformaldehyde, reagent grade, crystalline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">neutral-buffered formalin</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">72210</td>
			<td>10% neutral buffered formalin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">phosphate buffered saline (PBS)</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>10X phosphate buffered saline pH 7.4 (137mM NaCl, 2.5mM KCl, 4.3mM Na2HPO4, 1.4mM KH2PO4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml falcon tubes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">21008-216</td>
			<td>presterilized centrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">forceps</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">n/a</td>
			<td>fine forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chick saline&#160;</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a&#160;</td>
			<td>0.85% NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tinfoil</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paper towel</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought</td>
		</tr>
	</tbody>
</table>

organotypic culture method to study the development of embryonic chicken tissues

Embriyonik tavuk genellikle omurgalı geliştirme için bir güvenilir model organizma kullanılır. Onun erişilebilirlik ve kısa kuluçka dönemi yapar deneme için ideal. Şu anda, tavuk embriyo gelişimsel bu yollar çalışmanın inhibitörleri ve uyuşturucu yerelleştirilmiş sitelerde ve çeşitli yöntemler kullanarak düşük konsantrasyonlarda uygulayarak yapılır. Vitro doku kültürü, konak organizmanın aynı anda birçok fiziksel sınırlamalar mevcut embriyolar için duyarlılık gibi bütün embriyo ile çalışırken atlayarak sırasında ayrılmış dokuların çalışma sağlayan bir tekniktir ölümcül kimyasal yüksek dozda. Burada, kültür gelişimsel süreçleri ötesinde şu anda kurulan yöntemleri incelenmesi için yeni fırsatlar sunan embriyonik tavuk yarım baş vitro, kültür için protokol bir organotypic mevcut.

Bu yöntemi kullanarak, embriyonik tavuk gözün gelişimi (HH34) 8 günün vitro 4 gündür kültürlü. Dört gün içinde ovo gelişimi için HH38 karşılık gelir.
Bu kodlamayla Yöntem geliştirme tüy tomurcukları göz çevreleyen ve göz kapakları (Şekil 1B) destekler. Bu tüy tüy bud geliştirme kodlamayla döneminde başlatılan ovo içinde1...

Watch this Scientific Journal Video about Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues at JoVE.com

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Embriyonik tavuk doku gelişimi çalışmaya Organotypic kültür yöntemi

Burada, embriyonik tavuk organları büyümeye Protokolü kültür bir organotypic mevcut tüp bebek. Bu yöntemi kullanarak, embriyonik tavuk doku gelişimi, kültür ortamı üzerinde kontrol yüksek derecede korunarak okudu.

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

organotypic-culture-method-to-study-development-embryonic-chicken

Research

JoVE Journal

Developmental Biology

5.9K Görüntüleme.  Mount Saint Vincent University. Burada, embriyonik tavuk organları büyümeye Protokolü kültür bir organotypic mevcut tüp bebek. Bu yöntemi kullanarak, embriyonik tavuk doku gelişimi, kültür ortamı üzerinde kontrol yüksek derecede korunarak okudu.

Video: Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

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

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

Elektroporasyon tarafından Embriyonik Fare Koklear Eksplantlarından Kültür ve Gen Transferi

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

Gelişim Biyolojisi

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 ...

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 ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Embriyonik Kök Hücre ve Doku Kardiyak Farklılaşma epigenetik Yönetmelik

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

Bir roman<em&gt; Ex Ovo</em&gt; Yapıştırma Tekniği bir Embriyonik tavuk Sistemde İntrakardiyak hemodinamiği Alter

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

Fare Embriyonik metatarsallara Kültürü: endokondral Kemikleşmenin bir Fizyolojik Modeli

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

Fare Embriyonik Böbreklerinin Diseksiyonu ve Kültürü

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Çekirdeklerin dağıtım, proteinler ve sitoiskeleti çalışmaya Caenorhabditis elegans Germline sayısal analizi

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

Yalıtım embriyonik doku ve timus örnek kullanarak bıldırcın-tavuk Chimeric organ oluşumu

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

Embriyonik tavuk doku gelişimi çalışmaya Organotypic kültür yöntemi

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Elektroporasyon tarafından Embriyonik Fare Koklear Eksplantlarından Kültür ve Gen Transferi

Manipülasyon ve

Transcriptome Profil

Embriyonik Kök Hücre ve Doku Kardiyak Farklılaşma epigenetik Yönetmelik

Bir roman

Fare Embriyonik metatarsallara Kültürü: endokondral Kemikleşmenin bir Fizyolojik Modeli

Fare Embriyonik Böbreklerinin Diseksiyonu ve Kültürü

Çekirdeklerin dağıtım, proteinler ve sitoiskeleti çalışmaya Caenorhabditis elegans Germline sayısal analizi

Yalıtım embriyonik doku ve timus örnek kullanarak bıldırcın-tavuk Chimeric organ oluşumu

C. elegans Embriyonik Gelişimi Analiz Etmek Için Yarı-yüksek-throughput Görüntüleme Yöntemi ve Veri Görselleştirme Araç Seti