Bu araştırma [kısmen] Intramural araştırma NIH, Ulusal Enstitüsü Çevre Sağlık Bilimleri Program tarafından desteklenmiştir. İstatistiksel analiz için özel Shyamal Peddada teşekkürler. Biz de Humphrey Yao, Gary kuş ve Yuki Arao incelenmesi bu el yazması ve Franco St onun sürekli destek ve tavsiyeler için teşekkür etmek istiyorum.

Tüm işlemleri fare çekirdek dışarı NIEHS Knock tesisin normal çalışma kapsamında yapıldı. Bütün fareler bir 12:12 tarihinde tutulan s açık: koyu döngüsü ve bir diyet NIH-31 ve su besleme ad libitum. Tüm deneylerin hayvan bakım ve kullanım Komitesi, Ulusal Çevre Sağlık Bilimler Enstitüsü uygun olarak yapıldı.
1. hayvan hazırlık
<ol><li>Doğurmak donör fareler, B6 (Cg)-Tyr < c-2J > /J dişi fareler, 8-10 hafta-in yaş B6 için doğal olarak (Cg)-Tyr < c-2J > /J erkek fareler arasında 8 ve 26 hafta-in yaş. Fişler görsel olarak ertesi sabah (E0.5) kontrol edin.</li>
	<li>Alıcı fareler, İsviçreli kadın fareler 6 ve 9 hafta-in yaş arasında doğal olarak vasectomized İsviçre Webster erkek fareler için yetiştirilmiş Webster doğurmak. Fişler görsel olarak ertesi sabah (E0.5) kontrol edin.</li>
</ol>2. ES Hücre hazırlık
<ol><li>129-derieved AB2.2 ES hücreler için 15 ile desteklenmiş DMEM hücrelerde kültür % FBS, % 1 glutamin, %1 Penn/strep ve % 0.1 β-mercaptoethanol. ES Hücre tedarikçinin yönergeler, onların önerilen protokolleri kullanarak başına diğer tüm ES hücre kültür.</li>
</ol>3. embriyo koleksiyonu
<ol><li>
Hayvan diseksiyon
	<ol>
<li>Birkaç 60 mm yemekleri, yaklaşık 1 5 fare artı 10 mL patlama koleksiyonu medya (BCM) ile 3 ekstra hazırlamak (% 10 FBS inç DMEM), ve iyi bir 4 çanak ile 0.5 mL BCM de başına. 37 ° C, % 5 CO2equilibrate.</li>
		<li>B6 ötenazi (Cg)-Tyr < c-2J > /J dişi fareler, CO2 boğulma, % 10 debi ile kullanarak E3.5.</li>
		<li>Ötenazi sonra fareyi sırtüstü pozisyonda yerleştirin ve karın % 70 etanol ile iyice ıslak.</li>
		<li>Sadece, deri yoluyla ilk kesi kadar geniş açılış yapma karın, orta noktasını yaptırın. Sonraki yeniden yapım belgili tanımlık kesme mümkün olduğu kadar geniş karın duvarından açın. Not: iki adımda bunu azaltmak veya kürk kirlenme ortadan kaldırmak yardımcı olur.</li>
		<li>Yumurtalık, ovidukt ve rahim, yağ dokusu minimumda tutmak için ekstra bakım alarak bir yumurtalık başlangıç ve yumurtalık yağ yastığı künt forseps ile tutarak dikkatle çıkarın. Rahim bir tek birim olarak veya aşağıdaki yöntemde bir değişiklik ile bireysel boynuzları olarak kaldırın.</li>
		<li>Kaldırıldıktan sonra rahim patlama toplama ortamı içeren bir 60 mm çanak yerleştirin.</li>
	</ol>
</li>
	<li>
Embriyo kaldırma
	<ol>
<li>BCM ile dolu bir 10 mL şırınga hazırlamak ve bir 25 G iğne ile uygun.</li>
		<li>Her seferinde bir rahim BCM holding çanak çıkarıp, onları aşırı kan kaldırmak ve düşük bir büyütme diseksiyon mikroskop altında 60 mm tabak içinde yerleştirmek için bir doku üzerinde leke.</li>
		<li>Dikkatlice ve yavaşça rahim distal sonuna (ovidukt) hemen altında tutarak forseps diseksiyon ile paralel bir uçak mümkün olduğunca olarak iğnede rahmine ortasına yerleştirin. Forseps ipuçları ötesinde iğne yerleştirin.</li>
		<li>İğne için rahim tutulacağı pensler biraz baskı uygulamak ve yaklaşık 1 mL BCM ile rahim boynuz sınırdışı. Not: şırınga üzerinde çok fazla baskı medya, kontrol edilemeyen bir akışı neden olur bu adımda kez çanak bırakarak özen göstermelidir.</li>
		<li>Rahim, 60 mm çanak, başına en fazla 5 bütün uteri kızarma sonra çanak koleksiyonu kadar da kuluçka makinesine koyun.</li>
	</ol>
</li>
	<li>
Embriyo koleksiyonu
	<ol>
<li>Bir el çekilmiş Cam Pipet ve ağzını pipet cihazlar kullanarak, blastokistlerin 25 X ve 35 X arasında parçalanmış bir kapsam altında toplamak. (Şekil 1). Not: Ağız pipet aparatı 30-40 cm esnek boru oluşur, 5 mm altında kimlik eklemek bir 1 000 µL pipet ucu, ilk tek bir amaç için nokta ve içindeki Cam Pipet yapıştırmayın. Bir ekli 1 cc şırınga varil, bir ağız parçası hareket etmek hiçbir pistonu ile diğer ucunu bir şırınga filtre ekleyin.</li>
		<li>Bulaşık kuluçka makinesi--dan kaldırma ve bir ızgara deseni kullanılarak yemek embriyolar için her bölümünü inceleyin. Embriyo aldı tek tek yukarı ve yer üstünde BCM ikincil bir tabak içinde damla.</li>
		<li>Tüm yemekler toplama sonra embriyo bir kez daha taze BCM bir düşüş yıkama ve enjeksiyon beklemek üzere 4 iyi çanak içinde yerleştirin.</li>
	</ol>
</li>
</ol>4. embriyo enjeksiyon
<ol><li>ES hücreleri tek tek enjeksiyon iğneyi onayı ile seç.<ol>
<li>5-7 mL enjeksiyon medya ekleyerek bir enjeksiyon yemek hazırlamak (DMEM, %10 FBS ve 20 mM HEPES) bir cam alt enjeksiyon yemek için. İğne oda sıcaklığında yapılır.</li>
		<li>Bir hava veya yağ microinjector kullanarak, ES Hücre çizmek için enjeksiyon iğne vakum uygulanır. Hücreler arasında mümkün olduğunca az yer olarak tutmak için dikkat ediniz. Burada ya da enjeksiyon adım ES hücreleri saymak.</li>
		<li>Orta ve küçük boyutlu hücreler, pürüzsüz bir yüzey ile boşluklar gibi bariz hataları olmadan genel nüfusa göre ES hücreleri seçin. Not: ES Hücre popülasyonlarının büyük ölçüde morfolojik kalitesinde değişir ve kullanılan standartlar onlarla ayarlamanız gerekebilir.</li>
	</ol>
</li>
	<li>Embriyo yaklaşık 15 ES hücrelerle (Şekil 1) enjekte.<ol>
<li>Holding pipet eklemek ipucu için ikinci bir hava microinjector ve açılış sahibinin yakınındaki bir embriyo konumlandırın. Embriyo sahibi için güvenli için vakum uygulanır.</li>
		<li>Z ekseni için böylece embriyo sadece iyi zona üzerinde odaklanarak takip slayt, dokunuyor holding pipet yukarı veya aşağı taşıyarak ayarlayın. Ardından, ES hücrelerdir ucuna yakın odak enjeksiyon iğne Z ekseni ayarlayın.</li>
		<li>Embriyo şimdi hafifçe üst veya alt, ICM istediğiniz konuma koymak için dönen enjeksiyon iğne iterek ayarlayın.</li>
		<li>Zona ve ICM veya firma ama kontrollü push kullanarak trofoblast, enjeksiyon iğne yerleştirin. Microinjector enjeksiyon iğne blastocoel çöken düşük tutmak için hafif bir basınç uygulayın.</li>
		<li>Bir kez iğne blastocoel nüfuz, ES Hücre embriyo blastocoel için aktarmak için microinjector gelen basınç uygulayın. Basınç embriyo hücreleri tutarken, normale dönmek için izin vermek için embriyo çıkmak Duraklat kısaca iğne eğimi olarak başlar. Not: Başvuru grubu ES hücreleri normalde enjekte edildi, geleneksel bir ES takip enjeksiyon Protokolü9ICM dokunmamaya dikkat çekici, hücre. ES hücreleri de iğne doğrudan aracılığıyla ICM (TICM) (Şekil 2Tamamlayıcı film) iterek ICM yoluyla enjekte edildi.</li>
	</ol>
</li>
</ol>5. eEmbryos devri
<ol><li>Transfer embriyoların İsviçre Webster farelere, E2.5, üretim prosedürleri başına bir cerrahi olmayan embriyo transferi aygıtı kullanarak. Cerrahi olmayan bu yordam herhangi bir anestezi veya özel ameliyat sonrası bakım gerektirir. Tek tek embriyo ve teslim ve sonraki sütten kesme yoluyla transferden sonra evi fareler.</li>
</ol>

<ol>
	<li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Genome+Engineering+Using+CRISPR/Cas+Systems.">Multiplex Genome Engineering Using CRISPR/Cas Systems.</a> Science. 339 (6), 819-823 (2013).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6), 823-826 (2013).</li><li>Friedel, R., Seisenberger, C., Kaloff, C., Wurst, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EUCOMM+the+European+Conditional+Mouse+Mutagenesis+Program.">EUCOMM the European Conditional Mouse Mutagenesis Program.</a> Brief Funct Genomic Proteomic. 6 (3), 180-185 (2007).</li><li>Austin, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+knockout+mouse+project.">The knockout mouse project.</a> Nat Genet. 36 (9), 921-924 (2004).</li><li>Coleman, J. L., Brennan, K., Ngo, T., Balaji, P., Graham, R. M., Smith, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Knockout+and+Reporter+Mouse+Line+Generation+and+Breeding+Colony+Establishment+Using+EUCOMM+Conditional-Ready+Embryonic+Stem+Cells:+A+Case+Study.">Rapid Knockout and Reporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMM Conditional-Ready Embryonic Stem Cells: A Case Study.</a> Front Endocrinol (Lausanne). 30, 105 (2015).</li><li>Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., Roder, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+completely+cell+culture-derived+mice+from+early-passage+embryonic+stem+cells.">Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.</a> Proc Natl Acad Sci U S A. 90 (18), 8424-8428 (1993).</li><li>Eggan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+and+female+mice+derived+from+the+same+embryonic+stem+cell+clone+by+tetraploid+embryo+complementation.">Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation.</a> Nat Biotechnol. 20 (5), 455-459 (2002).</li><li>Eggan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+vigor,+fetal+overgrowth,+and+viability+of+mice+derived+by+nuclear+cloning+and+tetraploid+embryo+complementation.">Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.</a> Proc Natl Acad Sci U S A. 98 (11), 6209-6214 (2001).</li><li>Eakin, G. S., Hadjantonakis, A. K., Papaioannou, V. E., Behringer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+potential+and+behavior+of+tetraploid+cells+in+the+mouse+embryo.">Developmental potential and behavior of tetraploid cells in the mouse embryo.</a> Dev Biol. 288 (1), 150-159 (2005).</li><li>Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+production+of+mice+from+embryonic+stem+cells+injected+into+four-+or+eight-cell+embryos+by+piezo+micromanipulation.">Efficient production of mice from embryonic stem cells injected into four- or eight-cell embryos by piezo micromanipulation.</a> Stem Cells. 26 (7), 1883-1890 (2008).</li><li>Poueymirou, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F0+generation+mice+fully+derived+from+gene-targeted+embryonic+stem+cells+allowing+immediate+phenotypic+analyses.">F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses.</a> Nat Biotechnol. 25 (1), 91-99 (2007).</li><li>Behringer, R., Gertsenstein, M., Nagy, K. V., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse embryo. , (2014).</li><li>Pease, S., Saunders, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advanced Protocols for Animal Transgenesis. , (2011).</li><li>Young, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mechanical+stimulation+on+the+reprogramming+of+somatic+cells+into+human-induced+pluripotent+stem+cells.">Effects of mechanical stimulation on the reprogramming of somatic cells into human-induced pluripotent stem cells.</a> Stem Cell Res Ther. 8 (1), 139 (2017).</li></ol>

Yazarlar ifşa etmek rakip hiçbir ilgi alanlarına sahip.

Uzun ICM herhangi bir rahatsızlık ölüm, aslında, Şu anki blastosist mikroenjeksiyon protokolleri hala bu12,13uyarmak yol açabileceğini zannedildiği. Biz burada göstermiştir ne olduğunu mikroenjeksiyon ICM ile embriyo için zararlı değildir ve chimeras verimini artırır.
Böyle bir sonuç kesin mekanizması tanımlanmamış. Ancak, ICM ve eşlik eden hypoblast, mekanik bozulma ES Hücre entegrasyon mikroenjeksiyon takip y...

Yaklaşık 25 yıl için genetik olarak hedeflenen fareler oluşturulması genellikle bir darboğaz tarafından blastokistlerin ES Hücre mikroenjeksiyon aşamada chimeras verici germline üretimi için engel yavaş bir süreç olmuştur. Son gelişmeler, ES hücre ve yüksek üretilen iş ES hedefleme CRISPR/Cas91,2 de dahil olmak üzere hücre üretimi konsorsiyumlar KOMP gibi genetik olarak değiştirilmiş ES hücre üretimi/durumu düzeldi. Ancak, genetik olarak değiştirilmiş fare bu ES hücreleri3,4den oluştururken bir darboğaz germline chimeras nesil kalır. Yüksek işlem hacmi ES hücre üretimi projeleri germline chimeras başarılı oluşturulması için önemlidir ES Hücre kalitesi yüksek değişkenlik tarafından rahatsız olması. Ayrıca, yaygın olarak kullanılan ES Hücre satırlarından bazıları yüksek anöploidi ve germline yetkili chimeras5zor üretim için bilinir.
Fare ya da daha yüksek bir chimera oluşturmak için birçok yöntem geliştirilmiştir veya tamamen ES Hücre fareler6,7,8,9,10türetilmiş. Bu sistemlerin her biri kendi yararları olsa da, onlar da kendi eksiklikleri var. Verimsiz ve outbred satırları 5,6sınırlı iken % 100 ES Hücre üretme fareler, türetilmiş tetraploid chimeras kuşağıdır. Morula enjekte ederek yeni yöntemleri yüksek oranda chimeras, % 100, yaklaşan verim ama genel olarak önemli ek donanımları (lazerler ya da piezos) ve eğitim10,11gerektirir. Nerede bu teknikler zaten yerde laboratuvarlarda, bu yeni yöntem gerek kalmayabilir.
Bu çalışmanın amacı chimera üretme, sonraki fare koloni kurmak için mutant gen aktarımını germline olasılığını artırma oranı artırmak için ortak teknikleri ve hazır ekipman kullanan bir yöntem bulmak oldu.

<table><tbody><tr><td>ES Cell injection needle</td><td>Humagen</td><td>MSC-20-0</td><td></td></tr><tr><td>NSET device</td><td>Paratechs</td><td>60010</td><td></td></tr><tr><td>DMEM</td><td>Gibco (life technologies)</td><td>11965-092</td><td></td></tr><tr><td>FBS</td><td>Gibco (life technologies)</td><td>10439-024</td><td>lots should be individually tested for ES Cell compatibility.</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H0887</td><td></td></tr><tr><td>&amp;#946;-mercaptoethanol</td><td>Sigma</td><td>M7522</td><td></td></tr><tr><td>Micro manipulator</td><td>Leica</td><td>Micro manipulator</td><td></td></tr><tr><td>micro injector</td><td>Eppendorf AG</td><td>Cell Tram Air 5176</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>DM IRB</td><td></td></tr><tr><td>4 well dish</td><td>Thermo Scientific</td><td>176740</td><td></td></tr><tr><td>60 mm dish</td><td>Sarstedt</td><td>83.3901</td><td></td></tr><tr><td>B6(Cg)-Tyr&amp;#60;c-2J&amp;#62;/J</td><td>Jackson Labs, Bar Harbor, Maine, USA</td><td>000058</td><td></td></tr><tr><td>Swiss Webster mice</td><td>Taconic, USA</td><td>Tac:SW</td><td></td></tr><tr><td>Injection Dish</td><td>MatTek Corp.</td><td>P50G-0-30-F</td><td></td></tr></tbody></table>

trans-inner cell mass injection of embryonic stem cells leads to higher chimerism rates

Bir çaba ES Hücre metodolojileri ile genetik fareler oluşturulmasında verimliliğini artırmak için biz bir uyum geçerli blastosist enjeksiyon protokolü için mevcut. Burada basit bir rotasyon embriyo ve sızdırma Trans-iç hücre kütlesi (TICM) yoluyla chimeric % %31 50'si, hiçbir ek donanımları ile fareler yüzdesi arttı ya da daha fazla eğitim uzman raporu. 26 farklı klonlar yakın akraba evliliğinden doğan ve 35 toplam klonlar bir 9 ay boyunca enjekte edildi. Geleneksel enjeksiyon teknikleri ve TICM arasında anlamlı bir fark gebelik oranı veya embriyo iyileşme oranı oldu. Bu nedenle, enjeksiyon süreci ve blastosist basit bir konumlandırma ve ICM enjekte herhangi bir önemli değişiklik, ES Hücre blastocoel boşluğa bırakmadan potansiyel chimeric üretim ve sonraki miktar artırabilir germline iletim.

Hücre kültürünü rasgele etkisi olarak tedavi, karışık etkileri Doğrusal model (örneğin SAS (sürüm 9.2)) yazılımında verileri çözümlemek için kullanıldı. Her analiz embriyo transfer ve hücre sayısı için kontrollü. İkinci analiz de hayatta yavru sayısı için kontrol.
Bu çalışma için her ES hücre kültürünü ve klon her ikisi ile geleneksel enjekte ettiler ve TICM teknikleri, her embriyo sonra i...

Watch this Scientific Journal Video about Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates at JoVE.com

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

Burada yeni ekipman kullanmanıza gerek kalmadan chimera üretimi artırmak için bir iletişim kuralı mevcut. Enjeksiyon için embriyo basit yönlendirme değişikliği üretilen embriyo sayısını artırmak ve potansiyel olarak zaman çizelgesi germline iletim için azaltmak.

Trans-iç hücre embriyonik kök hücrelerin kitle enjeksiyon daha yüksek Chimerism oranları için yol açar.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

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10.2K Görüntüleme.  NIEHS/NIH. Burada yeni ekipman kullanmanıza gerek kalmadan chimera üretimi artırmak için bir iletişim kuralı mevcut. Enjeksiyon için embriyo basit yönlendirme değişikliği üretilen embriyo sayısını artırmak ve potansiyel olarak zaman çizelgesi germline iletim için azaltmak.

Video: Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Karşı Önceden tanımlanmış T Hücre Reseptör özgüllükleri Transnuclear Fare Toxoplasma gondii SCNT ile Elde Edilen

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

İmmünoloji ve Enfeksiyon

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Erken Postimplantation Fare Embriyolar içine Hücreleri veya DNA Kesinlikle lokalize Transferi Yöntemleri

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Gelişim Biyolojisi

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.

We describe a dramatically improved method for mouse cloning using trichostatin A, vitamin C, and deionized bovine serum albumin. We show a simplified, reproducible protocol that supports efficient development of cloned embryos. Hence, this method could become a standardized procedure for mouse cloning.

Kombinasyon tedavisi Trichostatin A ve C vitamini fareler somatik hücre nükleer Transfer tarafından klonlama daha etkili olmasını sağlar.

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Doku Hedeflenen Embriyonik Chimeras: Zebra balığı Gastrula Hücre Nakli

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Biyoloji

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

İntravenöz ve içi amniyotik rahim içinde nakli fare modeli

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Embriyonik Cardiomyocytes kuş embriyo implantasyonu Vivo için Mikroenjeksiyon tabanlı sistem

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

Chimeras Üretim Medaka Embriyolar ve Hücre Nakli Dechorionation

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

Tavuk embriyo haline İnsan Kök Hücre Xenotransplantation

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

Civciv Embriyolar Tek Hücreli Transfeksiyon

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

Genle Manipüle Edilmiş Fare Modelleri Geliştirmek için Embriyonik Kök Hücrelerde CRISPR / Cas9 Aracılı Yüksek Verimli Gen Hedefleme

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...