Biz Nancy Papalopulu ve Boyan BONEV Xenopus elektroporasyon ile yardım için teşekkür ediyoruz. Ayrıca, eleştirel okuma, Jeremy Green ve John Liu laboratuar verdikleri destek için yararlı tartışmalar ve üyeleri Wallingford Marc Dionne teşekkür ederim. Bu çalışma BBSRC (BB/E013872/1) ve Wellcome Trust (081880/Z/06/Z) KJL hibe tarafından finanse edildi.

Bölüm 1A. Ekipman Mikroskop: düşük güç amacı ile dik stereo diseksiyon kapsamı <ul><li> Gerilim / Pulse Generator: BTX ECM 830 Kare Dalga Elektroporasyon Sistemi </li><li> Pipet çektirmesi: P-87 Mikropipet Çektirme (Sutter Instrument Company, CA) </li><li> Manipulator: kaba, ya da kombine kaba ve ince hazırlık bağlı. </li><li> Mikropipet sahibi: Güzel Bilim Araçları </li><li> Elektrot: ev yapımı </li><li> Elektroporasyon kamara: ev yapımı </li></ul> L-şekilli elektrotlar: <ol><li> Yüksek saflıkta 8 cm 0.4 mm tungsten tel (Goodfellow) ve orta noktasında yapıştırmayın kesin 1ml şırınga kullanarak bir macun (Blu-Tack). 4 cm tungsten tel L şekline bend ucu ve şırınga ucu açık bırakın, ucundan 1 cm (Şekil 1A). </li><li> Ucu sonuna kadar uzunluğu 0.5 mm boyutlarında kesin. Tonun ucu elektrot uçtur. </li><li> Şırıngaya aşırı tungsten tel paralel ve elektrot nabız jeneratörü bağlamak için onu kullanabilirsiniz. </li><li> Bir çift elektrot verme işlemi tekrarlayın. </li><li> DC kablolar üzerinden kare dalga darbe jeneratörü elektrotları takın. </li></ol> Elektroporasyon kamara <ol><li> Hat alt ~ 5 mm non-toksik hamuru ile 90 mm çanak. </li><li> Elektroporasyon medya ile çanak doldurun. </li><li> No: 5 saatçilik forseps T-şekilli bir kuyu (Şekil 2) bölmek. ~ 2 mm x 2 mm x 10 mm ve kısa ~ 2 mm x 2 mm x 5 mm uzun iyi ölçmelidir. Elektroporasyon bulaşık yıkanır ve tekrar kullanılabilir. </li></ol> Bölüm 1B. Reaktifler DNA veya tahsil makromoleküllerin <ul><li> Mikropipetler: 1 mm genişliğinde 4 &quot;uzun borosilikat cam kapilerleri (TEFE, TW100-F) </li><li> Kültür medya: Normal Amfibi Medya (NAM) </li><li> &gt; 10 X stoğu: 1100 mM NaCl, 20mM KCL, 10 mM Ca (NO 3) 2 • 4H 2 O, 1 mM EDTA. <ul><li> 4 ° C&#39;de Otoklav ve mağaza </li><li> 1 X NAM: 10x stok, tampon 0.1 mM NaHCO 3 ve 0.2 mM Na 3 PO 4 ile seyreltilir. </li></ul></li><li> Xenopus laevis tetarlar, sahne 28 </li></ul> DNA hazırlığı: <ol><li> Ifade plazmid standart protokolleri kullanarak hazırlayın. </li><li> Nükleaz serbest H 2 0 1 mcg / ml final konsantrasyonda DNA tekrar </li></ol> * PCS2 gibi güçlü bir CMV organizatörü içeren vektörler, başarı vardı + [16]. Soy analizi için, biz genellikle son bir 0.1 mg / ml konsantrasyonda yeşil floresan proteininin (pCS2 GFP) kodlayan DNA içerir. [0.1-3 mg / ml arasında DNA konsantrasyonları da test edildi. Biz bulduğu konsantrasyonları 0.8 mg / ml verimsiz etiketli hücre altında, daha fazla DNA konsantrasyonları ise 2 mg / mu; l elektroporasyon verimliliğini artırmak vermedi]. Morfolino oligonükleotid hazırlanması: (Not:. Pratisyenler fluoresceinated gerekir (3&#39;-carboxyfluorescein değiştirilmiş) veya başka bir ücret) <ol><li> Süspanse edin morfolino oligonükleotidler (MOS) (Genetools, <a href="http://www.genetools.com" target="_blank">www.genetools.com</a> nükleaz serbest H 2 0 2 mM konsantrasyonda). </li><li> Isı kısım stok solüsyonu 65 ° C&#39;de 5 dakika. </li><li> Son konsantrasyonu 0.5 mM nükleaz ücretsiz su sulandırınız. </li></ol> * 0.1-1mm MO çözümleri test edildi. 0.5 mM MO çözümler birçok mezenkimal hücreler elektroporasyon için yeterli oldu. Mikropipetler <ol><li> Borosilikat cam kılcal damarlar (1 mm genişliğinde, 4 &quot;uzun, TEFE yok. TW100-F) mikropipetler hazırlayın. Mikropipetler hazırlamak, 8-12 mm uzunluğunda konik ve ince uçlu iğne çektirmesi kullanın. </li><li> Crush ucu ~ 2 mmucundan, forseps kullanarak pürüzlü bir duraklama. </li></ol> Medya <ol><li> Kuluçka medya: 1x stok taze 3 / 4 Normal Amfibi Medya (NAM) hazırlayın. 0.025 mg / ml gentamisin ekleyin. </li><li> Elektroporasyon medya: yukarıdaki gibi,% 0.1 Benzocaine (Sigma, 06.950). </li></ol> 2. Elektroporasyon Mikropipet kurulum <ol><li> Mikropipet ~ 1 ul enjeksiyon solüsyonu ile doldurun. </li><li> Mikromanipülatör mikropipet Güvenli ve mikroenjektör (Picospritzer II) ekleyin. </li><li> Masa üstü ile 50 ° açı mikropipet. </li><li> Enjeksiyon basıncı 20 PSI olarak ayarlayın. </li><li> Puls başına 30 nl enjekte mikropipet kalibre edin. </li></ol> Tadpole hazırlık <ol><li> 5 dakika elektroporasyon medya kuluçka aşamasında 28 Xenopus larvaları anestezisi. </li><li> Elektroporasyon medya ile dolu elektroporasyon odasına anestezi larva transferi. Pozisyonu embriyoUzun içinde baş, aşağı dorsal tarafına ve ventral tarafta maruz T kavşak aittir. Kafa, kuyruk göre biraz yüksek olmalıdır. </li><li> Forseps kullanarak, hafifçe hamuru çevreleyen ile larva güvenli. (Not:. Larva güvenli değilse, seğirme ve elektroporasyon sırasında elektrot ile irtibata Bu durumda yüz dokular ciddi hasar görecek olması, tetra atmak) </li></ol> Elektroporasyon <ol><li> Mikropipet ucu hemen çimento bezi ve arka yüz mezenşimin içine yerleştirin. </li><li> 30 nl çözüm mezenşimin içine enjekte edilir. </li><li> Mikropipet geri çekin. </li><li> Hızla embriyonun kafası (Şekil 3) paralel elektrot uçları aynı hizaya getirin. </li><li> 8 50 ms, 20mV kare darbeleri uygulayın. </li><li> Elektrotlar geri çekin. </li><li> Forseps kullanarak dikkatli bir şekilde larva serbest ve 3 / 4, NAM, 0.025 mg / ml gentamisin transfer. </li><li> 3 / 4, NAM, 0.025 mg / ml overn tetarlar inkübe edilmelidir.ight, ya da daha uzun. </li><li> 24 saat sonra floresan mikroskobu ile verimli elektroporasyon Ekran embriyolar. </li></ol> 3. Temsilcisi Sonuçlar: Floresan moleküllerin electroporated embriyoların kullanımı kolay tarama sağlar. Şekil 4 14.5 inkübe MO electroporated tetarlar ~ 12, 48 ve 96 saat sonra elektroporasyon tipik bir parti, ° C Floresan mikroskobu kullanarak, katille elektroporasyon sonra hemen görüntülenebilmekte ve elektroporasyon sonra birkaç gün süreyle devam edilebilir. Bizim tecrübelerimize göre, floresan aşamada 46 (~ 5 gün sonra) zayıf açıktır. Kıkırdaklar, floresan farklılaşma (~ St 42) başladıktan sonra dramatik bir şekilde azalır, ancak MO floresan gibi faringeal endoderm gibi diğer hücre tiplerinde daha güçlü devam. Floresan mikroskopi oligonükleotidler kıkırdak dahil olmak üzere birçok kraniyofasiyal dokular dahil olduğunu gösterir. Oligonükleotid floresans cgenellikle başın iki tarafında doku görüntülenebilmekte. Bu büyük olasılıkla elektroporasyon önce gevşek kraniyofasiyal mezenşimin boyunca enjeksiyon solüsyonu hızlı difüzyon nedeniyle. <img alt="Şekil 1" src="/files/ftp_upload/3381/3381fig1.jpg" /> Şekil 1 Homemade elektrotlar. L-şekilli tungsten tel, toksik olmayan kil veya macun kullanarak 1 ml şırınga eklenir. (A) elektrot terminus, 5 mm. (B) Termini paralel olarak, bir çift elektrot takın. Elektrotlar DC kabloları ile darbe jeneratörüne bağlı. <img alt="Şekil 2" src="/files/ftp_upload/3381/3381fig2.jpg" /> Şekil 2 Elektroporasyon kamara. (A) hamuru ile kaplı 90 mm çanak medya ve No 5 saatçilik forseps ile oyulmuş bir T-şeklindeki haznesi ile doludur. (B) uzun kenarı 2 mm X 2 mm X10 mm boyutlarındakısa önlemleri 2 mm X 2 mm x 5 mm iken. Embriyonun baş T-kavşak, ventral tarafta aittir. <img alt="Şekil 3" src="/files/ftp_upload/3381/3381fig3.jpg" /> Şekil 3 şematik gösteren elektroporasyon prosedür. St 28 larva ventral yüzü yukarı, elektroporasyon odasında yerleştirilir. Mikropipet yüz mezenşimin yatan çimento bezi içine yerleştirilir. Enjekte edilir. Mikropipet kaldırılır ve L-şekilli elektrotlar kafa paralel yan hizalanır. Sekiz 50 ms, 20 mV kare darbeleri uygulayın. Elektrotlar geri çekin. Istenilen safhalarında tetarlar büyütün. Pratisyenler veya floresan mikroskobu kullanarak GFP ifade görselleştirin. <img alt="Şekil 4" src="/files/ftp_upload/3381/3381fig4.jpg" /> Şekil 4 Temsilcisi tetarlar 12 (A), 48 (B) ve 96 (C) saat elektroporasyon (sırasıyla aşamaları 30, 34 ve 44). (A-B &quot;) Floresan MO geyik kraniyofasiyal mezenşimin içinde görüntülenebilmektees 30 ve 34. Floresans aşamada 44 (ok ucu, C&#39;-C &quot;) az kıkırdaklar tespit edilebilir. Gut yüksek autofluorescent.

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Yazarlar herhangi bir çıkar çatışması var.

Bu video, Xenopus iribaşların yüz mezenşimin elektroporasyon aracılı gen teslim fizibilite göstermiştir. Bu yaklaşımı kullanarak, bize daha sonraki zaman noktalarında belirli dokuları hedef sağlayan genin fonksiyonu manipüle erken gelişimsel etkileri atlayabilir. Çalışmalar bize electroporated hücrelerin yanı sıra ilgi proteinlerin hücre özerk gereksinimleri soy incelemek için izin, heterojen toplumlarda kraniyofasiyal mezenkimal hücreler etkilenmiş olabileceğini göstermektedir. Canlı görüntüleme ile birlikte, biz kraniyofasiyal gelişimi sırasında, zaman içinde gen fonksiyonunu incelemek için bu yaklaşımı kullanabilirsiniz. Bu yeni bir yöntem organogenez çalışma Xenopus tractability vurgulamaktadır. Biz genel olarak bu yöntem de morfolojilerinden ve diğer dokulara farklılaşma çalışma adapte edilebilir olduğunu tahmin ediyoruz.

A correction was made to <a href="http://http://www.jove.com/video/3381">Electroporation of Craniofacial Mesenchyme</a>. There was an incorrect unit used for the voltage.

Erratum: Electroporation of Craniofacial Mesenchyme

electroporation of craniofacial mesenchyme

Elektroporasyon, hassas zaman noktalarında dokulara ve hassas yerlerde DNA ve diğer ücret makromoleküllerin teslim etkili bir yöntemdir. Örneğin, elektroporasyon Xenopus sinir ve retina gelişimini incelemek için büyük bir başarı ile kullanılan, tavuk ve fare 1-10. Ancak, tüm bu çalışmalarda, araştırmacılar yumuşak dokuların hedefleyen çalışmalar olduğunu not etmek önemlidir. Kraniyofasiyal gelişim ile ilgilenen, çünkü biz bir yöntem yüz mezenşimin hedef uyarladı. Edebiyat aradı, biz, bizim için sürpriz, kıkırdak dokusu içine başarılı gen transferi çok az raporlar bulundu. Bu çalışmaların çoğu, gen terapisi çalışmaları, chondrogenic hücre hatları içine siRNA veya protein teslimat, ya da artrit 11-13 hayvan modelleri gibi . , Tavuk ya da fare gibi diğer sistemleri, yüz mezenşimin elektroporasyon zorlu olmuştur (kişisel HABERLEiyonlar, Kraniofasial Geliştirme Bölümü, KCL). Biz Xenopus procartilaginous ve kıkırdak dokulara elektroporasyon daha iyi çalışması olabileceği varsayımında bulunduk. Çalışmalarda, biz yüz kıkırdaklar gen transferi yüz primordium hala kıkırdak farklılaşması önce yumuşak doku oluşur erken evrelerinde (28), verimli bir şekilde oluşur. Xenopus kraniyofasiyal gelişim analizi için çok erişilebilir omurgalı sistemi. Xenopus embriyoların en erken aşamalarında analizler sağlayan, dışarıdan döllenmiş ve canlı tek bir hücre çözünürlükte görüntüleme yanı sıra annelere 14 yeniden kolaylaştırılması Kraniofasial yapıları nedeniyle daha kolay görünür Xenopus başka herhangi bir omurgalı modeli daha, öncelikle. Dışarıdan gelişmekte olan omurgalı modeller arasında, Xenopus larvaları di daha büyük ve daha kolay olarak Xenopus zebrafish daha kraniyofasiyal analizi için daha yararlı .ssect ve gelişmekte olan yüz bölgesine balık eşdeğer bölgesi dışında görüntüleme için daha erişilebilir. Buna ek olarak, Xenopus zebrafish (~ 100 milyon yıl yakın) 15 daha insanlar için evrimsel olarak yakın. Son olarak, bu aşamada, Xenopus tetarlar şeffaf ve floresan proteinlerle veya moleküller eşzamanlı ifade gelişmekte olan kıkırdaklar daha kolay görsellik sağlayacaktır. Biz bu yaklaşımı bize daha hızlı ve verimli bir şekilde in vivo model sistem bir aday molekülleri test etmek için izin olacağını tahmin ediyoruz.

Watch this Scientific Journal Video about Electroporation of Craniofacial Mesenchyme at JoVE.com

Electroporation of Craniofacial Mesenchyme

Kraniofasial kıkırdaklar diğer dokuları ile yakın temas içinde gelişir ve canlı hayvan işlemek için zordur. Elektroporasyon erken embriyonik etkileri atlayarak kraniyofasiyal iskeletin büyüme sırasında moleküler araçlar sunmak için kullanıyor. Bu yaklaşım, aday moleküllerin etkin bir şekilde test etmek için bize izin verecek<em&gt; In vivo.</em

Kraniofasial mezenşimin, Elektroporasyon

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

electroporation-of-craniofacial-mesenchyme

Research

JoVE Journal

Biology

11.8K Görüntüleme.  King's College London. Kraniofasial kıkırdaklar diğer dokuları ile yakın temas içinde gelişir ve canlı hayvan işlemek için zordur. Elektroporasyon erken embriyonik etkileri atlayarak kraniyofasiyal iskeletin büyüme sırasında moleküler araçlar sunmak için kullanıyor. Bu yaklaşım, aday moleküllerin etkin bir şekilde test etmek için bize izin verecek In vivo.

Video: Electroporation of Craniofacial Mesenchyme

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Mikobakteriler, Elektroporasyon

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

Biyoloji

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

Kraniofasial Geliştirme sırasında Ektodermal Epiteli Sinyal Özellikleri değerlendirilmesi

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

Embriyonik Chick Retina Ovo Elektroporasyon

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Kranial mesenchyme Hücresel Davranış Değerlendirme için bir Eksplantasyon Assay

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Genom çapında Gen Delesyonlar içinde<em&gt; Streptococcus sanguinis</em&gt; Yüksek Verimli PCR ile

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

Fare Embriyonik Yüz Ektoderm ve mesenchyme ayrılması

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Sox10 içinde Kraniyofasiyal Kalkınma Görselleştirme: kaede Transgenik Zebra balığı Hattı Time-lapse konfokal mikroskopi kullanılarak

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

4D konfokal mikroskopi kullanılarak Zebra balığı Kranyofasiyel morfogenez Analizi

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

İnsan Pluripotent için alternatif Kültürleri Hücre Üretim, Bakım ve Genetik Analizi Kök

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

Bıldırcın-ördek Chimeras kullanma Kraniyofasiyal Kalkınma için Türlere özgü Katılımlar değerlendirilmesi

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Methods Article

Kraniofasial mezenşimin, Elektroporasyon