Yazarlar Şekil 1 ve yardım ve tartışma için laboratuvarın diğer üyeleri embriyonun kafaları göstermek için Irene Choi teşekkür etmek istiyorum. Bu çalışma NIH hibe DE012728 (TW) tarafından desteklenmektedir

1. Dispase II hazırlayın <ol><li> Hazırlama taze Hepes-tamponlu salin (HBS) (50 mM Hepes / KOH pH 7.4, 150 mM NaCl). 16.8 ml Ultra Saf H 2 O içine 2 ml 0,5 M Hepes / KOH pH 7.4 ve 1.2 ml 2.5 M NaCl ekle </li><li> 10 mg / ml dispase II olun. 0.2 g dispase II (Roche, Kedi # 4942078001) tartılır ve 20 ml HBS içinde çözülür. Uzun süreli depolama için -20 ° C&#39;de bireysel Eppendorf tüpleri ve mağaza içinde 1.0 ml alikotları hazırlayın. </li><li> Kullanımdan önce PBS içinde 10 mg / ml dispase II 1:10 seyreltin. Buz üzerinde seyreltilmiş dispase II koyun. </li></ol> 2. Hamile Kadın CD-1 E10.5 Embriyolar teşrih Tüm hayvan deneyleri Colorado Denver Üniversitesi (UCD) Hayvan Bakımı ve Kullanımı Komitesi tarafından onaylanan protokoller çerçevesinde yapıldı. CD-1 (ICR) fareler Harlan Laboratories (Indianapolis, IN) elde edildi. <ol><li> % 70 etanol ile diseksiyon alanı ve araçları temizleyiniz. </li><li> Approv kullanarak hamile CD-1 kadın Euthanizeed protokolleri. </li><li> % 70 etanol ile karın püskürtün sonra karın açın. Rahim mesometrium uzakta gözyaşı rahim ve başka bir çekmek için forseps bir set kullanın. Gövde, uterus çıkarın. Karın diğer tarafında uterin horn için tekrarlayın. </li><li> Kısaca kan yıkamak için 10 cm Petri kabındaki buz PBS ile rahim durulayın. </li><li> Buz gibi soğuk PBS ile bir yeni bir Petri kabı içine rahim aktarın. Implantasyon siteler arasında keserek tek embriyo parçalara ayırın. </li><li> Bir stereomikroskop altında buz gibi soğuk PBS içine bir embriyo transfer. Yolk kesesi ile embriyonun maruz kesim sitelerinden birini başlayan rahim kas duvarı yırttınız ince forseps kullanın. Yolk kesesi ve amniyon soyun. Buz PBS ile 6 cm Petri kabı disseke embriyo transfer. Tüm embriyolar bu şekilde toplanan edilene kadar devam edin. </li></ol> 3. Dispase II Tedavisi <ol><li> 6-santimetrelik bir petri tabağına PBS ile tüm embriyolar yıkayın. DeğiştirmekPBS içinde seyreltilmiş 10 mL dispase II ile PBS ile yıkandı. 25 dakika boyunca 37 ° C&#39;de Petri kabı ve inkübe Kapak - bakteriyel bir inkübatör veya sıcak bir odada yeterli olmalıdır - bu adım için bir CO 2 doku kültürü inkübatör kullanmadım. </li><li> Petri kabı çıkarın ve bir stereomikroskop altında embriyo gözlemlemek. Ektoderm gevşek olması gerektiği, bu doku tabakası ve altta yatan mezenşimi arasında açık bir uçurum tiplemesindeki. Aksi takdirde, 37 bir ek 5 dakika ° C de inkübe Sonra buz üzerinde Petri kabı koyun. </li></ol> 4. Bozulmamış Yüz çıkıntıların üzerinden teşrih <ol><li> (VWR, Kedi # 14.672-380) atılabilir cam pipet kullanın Bir stereomikroskop altında bir dispase II 6-santimetrelik bir petri tabağına buz gibi soğuk PBS içine embriyo tedavi aktarmak için. </li><li> Embriyo tutmak için forseps bir çift kullanın. Kafa ve yüz çıkıntıların sınırları (Şekil 1 sınırlarını göstermektedir kesmek için dikkatli bir şekilde ince forseps bir çift kullanımıyüz çıkıntıları). Bir MNP bir tarafında, daha sonra MXP, ve son olarak FNP başlatın. Bozulmamış üzerinden yüz çıkıntıları (Şekil 2A) incelemek için diğer tarafa aynı işlemi uygulayın. </li></ol> 5. Mesenchyme Ayrı Yüz Ektoderm <ol><li> 3x çalışma mesafesi ile bir stereomikroskop altında yüz çıkıntıları gözlemleyin. Hafifçe yukarı ve aşağı 5 kez yüz çıkıntıları Pipeti uzun bir cam Pasteur pipeti kullanın. Bundan sonra, ektoderm soyulmasına kolaydır. </li><li> Yüz çıkıntıları tutun forseps bir çift kullanın. Çok yavaş ektoderm kalkmasına forseps diğer çifti kullanın. Şekil 2B yüz ektoderm ayrılmış sayfaları gösterir. </li><li> Buz PBS ile bir 1.5 ml Eppendorf tüp içine yüz ektoderm aktarmak için yeni bir uzun cam Pasteur pipeti kullanın. Buz PBS ile başka bir 1.5 ml Eppendorf tüpüne mezenşimi aktarın. </li><li> Her diseksiyon için yeni buz soğuk PBS kullanın. Örnekleri toplayın. </li><li> T yıkayınPBS ile o örnekleri. 4 ° C, 3 dakika boyunca 500 xg&#39;de santrifüjleyin. </li><li> Uzun cam Pasteur pipetler kullanarak PBS aspire. Örnekler ChIP tahlil için kullanılacak ise, 6.1 adım devam edin. RNA ekstraksiyonu için 6.2 adıma geçin. Protein ekstraksiyonu için 6.3 adıma geçin. </li></ol> 6. Daha Deney için Proses Örnekleri <ol><li> ChIP tahlil için örnekler <ol><li> Çapraz bağ ile% 1 formaldehit ile 1 ml PBS ekleyin. Örnekler homojenize edilir. </li><li> Rotasyon ile 10 dakika süreyle oda sıcaklığında inkübe edin. </li><li> Tepkimeye girmemiş formaldehit gidermek için her tüpe 1.25 M Glisin 110 ul ekleyin. </li><li> Karıştırılır ve 5 dakika boyunca oda sıcaklığında inkübe edilir. </li><li> 3 dk için 4 ° C sıcaklıkta 500 x g&#39;de Spin. Süpernatantı. </li><li> Dokulara yıkamak için 1 ml soğuk PBS ekleyin. </li><li> 3 dk için 4 ° C sıcaklıkta 500 x g&#39;de Spin. </li><li> PBS çıkarın ve PBS kez yıkayın tekrarlayın. </li><li> Süpernatantı, sıvı azot içinde doku dondurma oturtun. Sa Mağaza-80 ° C daha sonraki havuzu ve işlenmesi için mples. </li></ol></li><li> RNA ekstraksiyonu için Örnekler <ol><li> RNAlater (Ambion) ihtiva eden tüplerde yüz ektoderm ve mezenşimi yerleştirin. </li><li> ° C doku tam nüfuz izin vermek için gece boyunca 4 RNAlater Çözelti, numuneyi inkübe edin. </li><li> Sonraki havuzu ve işlenmesi için -20 ° C&#39;de saklayın. </li></ol></li><li> Protein ekstraksiyonu için Örnekler </li></ol> Sonraki işlemler için örnek lizis tamponu ekleyin. Veya sıvı azot içinde doku dondurma oturtun. -80 ° C daha sonraki havuzu ve işlenmesi için örnekler saklayın. Notlar <ol><li> Zaman kısıtlamaları. Yoluyla diseksiyon baştan Bölüm 6 başlangıcına 4-6 saat üzerinde planlayın. </li><li> Zaman yetkin, bu bölümleri 4 ve 5 (öne diseksiyon ve ektoderm ayırma) gerçekleştirmek için embriyo başına ~ 10-15 dakika sürer. </li><li> Örnekleri RNA izolasyonu, her eriyik için isen ilk Diethylpyrocarbonate ile tedavi edilmelidir. Ayrıca forseps and Away RNaz ile çalışma alanı temizleyin. </li><li> Dispase II tedavi ve diseksiyonu dışında her zaman buz üzerinde örnekler tutun. Diseksiyon için kullanılan PBS soğuk olduğundan emin olmak için her diseksiyon için yeni buz PBS değiştirin. </li><li> Sharp forseps hassas diseksiyon elde etmek için önemlidir. Herhangi bir bulaşmayı önlemek için tamamen forseps temizleyin. </li><li> Farklı çıkıntıların örnekler toplamak için dispase II tedaviden sonra ayrı ayrı öne teşrih. Sonra ektoderm ve mezenşimi ayırın. </li><li> Dispase II ile tedaviden sonra, yüz ektoderm soyulmasına kolaydır. Diğer ektodermden kontaminasyon özellikle dikkat edin. Tüm potansiyel olarak kontamine doku atın. </li><li> Sonra örnekleri 6. Bölümde uygun noktaya ulaşana kadar durmak yok, başlar. Yüz ektoderm ayrılması bitirin ve elinizden geldiğince hızlı olarak tüm embriyolar için mezenşimin. </li><li> Edispase II nzyme faaliyetlerini gruplar ve markalar arasında farklı olabilir. Farklı marka ve dispase II partiler için tedavi süresini ayarlamak için gereklidir. </li></ol>

<ol>
	<li>Wilkie, A. O., Morriss-Kay, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+craniofacial+development+and+malformation.">Genetics of craniofacial development and malformation.</a> Nat. Rev. Genet. 2, 458-468 (2001).</li><li>Gritli-Linde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2+-+The+Etiopathogenesis+of+cleft+lip+and+cleft+palate:+usefulness+and+caveats+of+mouse+models.">Chapter 2 - The Etiopathogenesis of cleft lip and cleft palate: usefulness and caveats of mouse models.</a> Current Topics in Developmental Biology. 84, 37-138 (2008).</li><li>Schutte, B. C., Murray, 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=The+many+faces+and+factors+of+orofacial+clefts.">The many faces and factors of orofacial clefts.</a> Human Molecular Genetics. 8, 1-7 (1999).</li><li>Chai, Y., Maxson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+craniofacial+morphogenesis.">Recent advances in craniofacial morphogenesis.</a> Dev. Dyn. 235, 2353-2375 (2006).</li><li>Jiang, R., Bush, J. O., Lidral, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+upper+lip:+Morphogenetic+and+molecular+mechanisms.">Development of the upper lip: Morphogenetic and molecular mechanisms.</a> Dev. Dyn. 235, 1152-1166 (2006).</li><li>Reid, B. S., Yang, H., Melvin, V. S., Taketo, M. M., Williams, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectodermal+WNT/&#946;-catenin+signaling+shapes+the+mouse+face.">Ectodermal WNT/&#946;-catenin signaling shapes the mouse face.</a> Developmental Biology. 349, 261-269 (2011).</li><li>Feng, W., 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=Spatial+and+temporal+analysis+of+gene+expression+during+growth+and+fusion+of+the+mouse+facial+prominences.">Spatial and temporal analysis of gene expression during growth and fusion of the mouse facial prominences.</a> PLoS ONE. 4, e8066 (2009).</li><li>Chia, R., Achilli, F., Festing, M. F., Fisher, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+uses+of+mouse+outbred+stocks.">The origins and uses of mouse outbred stocks.</a> Nat. Genet. 37 (11), 1181-1186 (2005).</li><li>Nagai, T., Aruga, J., Takada, S., 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+expression+of+the+mouse+Zic1,+Zic2,+and+Zic3+gene+suggests+an+essential+role+for+Zic+genes+in+body+pattern+formation.">The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation.</a> Developmental Biology. 182, 299-313 (1997).</li><li>Britsch, S., 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+transcription+factor+Sox10+is+a+key+regulator+of+peripheral+glial+development.">The transcription factor Sox10 is a key regulator of peripheral glial development.</a> Genes Dev. 15 (1), 66-78 (2001).</li></ol>

Yazarlar herhangi bir çıkar çatışması veya bu makalenin içeriği ile ilişkili diğer çatışmalar var.

Bu protokol, bir ilk dispase II işlenme adımını esas fare embriyonik yüz ektoderm ve mezenşim ayırmak için basit bir yöntem sağlamaktadır. Daha önceki çalışmalarda, önceden dispase II tedavi yüz çıkıntıları diseksiyonu uygulandı, ama biz sürekli mezenşimi &quot;yapışkan&quot; ve işlemek için daha zor hale gelmesidir bulduk. Bizim yeni protokol bu sorunu önler. Kombinasyon dispase II aşağı yukarı pipetleme ve tarafından nazik fiziksel gücü ile tedavi ektoderm ve kolay mezenşimi ayrı...

<table>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <td>Dispase II (neutral protease, grade II)</td>
 <td>Roche</td>
 <td>4942078001</td>
 <td>Make 10 mg/ml Dispase II stock solution in HBS. Aliquot and store at -20 &deg;C . </td>
 </tr>
 <td>RNAlater</td>
 <td>Ambion</td>
 <td>AM7021</td>
 <td>&nbsp;</td>
 </tr>
</table>

separation of mouse embryonic facial ectoderm and mesenchyme

Orofasiyal yarıkların 1000 bebekten dünya çapında 1,2 1,5 etkileyen en sık kraniofasiyal defektlerin vardır. Orofasiyal yarıklaşmanın anormal yüz gelişme 3 neden olur. Yüz insan ve fare, ilk büyüme ve desenlendirme içindeki doku birkaç küçük tomurcukları, 4,5 yüz çıkıntıları dayanır. Eşleştirilmiş frontal burun süreçler (FNP), maksiller çıkıntıların (MXP) ve mandibular çıkıntıların (MdP): yüz altı ana çıkıntıların türetilmiştir. Bu çıkıntılar bir örten epitel kaplı bulunmaktadır mezenşimi ve şişlikler oluşur. Birden fazla tür çalışmalar yüz ektoderm ve mezenşimi arasındaki karışma sinyal yüzünü 6 şekillendirme için kritik olduğunu göstermiştir. Ancak, bu sinyal röleleri Genlerdeki ilgili mekanistik ayrıntıları eksiktir. Bir gen ekspresyonu kapsamlı bir anlayış kazanmak için bir yol, bağlayıcı transkripsiyon faktörü ve develo ile ilişkili kromatin işaretleriping yüz ektoderm ve mezenşimi ayrılmış doku kompartmanları izole ve karakterize etmektir. Burada embriyonik gün (E) 10.5, çıkıntıların füzyon öncesinde fare yüz oluşumunda kritik bir gelişimsel aşamada yüz ektoderm ve mezenşimi ayırmak için bir yöntem mevcut. Bizim yöntemi daha önce yüz çıkıntıları 7 diseksiyon için kullanmış yaklaşımı uyarlanmıştır. Bu önceki çalışmada kendilenmiş C57BL istihdam vardı / Bu zorlanma gibi 6 farelerin genetik, genomik ve yüz morfolojisi 8 için bir standart haline gelmiştir. Burada, buna rağmen, uygun bir doku daha sınırlı miktarlarda nedeniyle, biz hayvancılık için, daha sağlam satın almak daha ucuzdur outbred CD-1 suşu kullanılmıştır ve herhangi kendilenmiş daha çöp başına daha fazla embriyo (12-18) üretmek eğilimi var fare zorlanma 8. Embriyo izolasyon takiben, nötr proteaz dispase II bütün embriyo tedavi etmek için kullanılmıştır. Sonra, yüz çıkıntıları teşrih edildied dışarı ve yüz ektodermden mezenşimi ayrıldı. Bu yöntem, yüz ektoderm ve mezenşim her ikisi de bozulmadan muhafaza eder. Bu yöntemi kullanarak elde edilen örneklerin protein tespiti, kromatin immünopresipitasyon (ChIP) tayini, mikroarray çalışmalar ve RNA-seq dahil teknikleri kullanılabilir.

Dispase II Tedaviden sonra, embriyonun ektoderm gevşek olma eğilimindedir. Yüz çıkıntıları diseksiyonu (Şekil 2A) sonra sağlam olmalıdır. İzole yüz ektoderm net ve mezenşimi doku (Şekil 2B) ücretsizdir. Protokolünün etkinliğini belirlemek için ve biz önbeyin için tahlil çapraz kontaminasyon tespit Zic3 9, ektodermal belirli cdh1 5 ve ters transkriptaz PCR kullanarak mezenkimal belirli sox10 10 dile getirdi. Çalışmalarımız yü...

Watch this Scientific Journal Video about Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme at JoVE.com

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Embriyo yüz ektoderm ve mezenşimi ayrılması için bir protokol tanımlanmıştır. Biz, ilk bütün embriyolar tedavisinde tüm yüz çıkıntıları üzerinden incelemek dispase II kullanın ve sonra yüz ektoderm ve mezenşimi ayırın.

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 ...

separation-of-mouse-embryonic-facial-ectoderm-and-mesenchyme

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JoVE Journal

Biology

10.8K Görüntüleme.  University of Colorado Denver Anschutz Medical Campus. Embriyo yüz ektoderm ve mezenşimi ayrılması için bir protokol tanımlanmıştır. Biz, ilk bütün embriyolar tedavisinde tüm yüz çıkıntıları üzerinden incelemek dispase II kullanın ve sonra yüz ektoderm ve mezenşimi ayırın.

Video: Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Proteinlerin Elektrogöçer Ayırma

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Biyoloji

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Fare Embriyonik Germinal Hücreleri İzolasyon ve türetilmesi

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo Görüntüleme ve Yumurtalık Kanseri Ortotopik Fare Modeli Tedavi Tedaviler

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

Kültür İlköğretim Fare Embriyonik Fibroblastlar Sinyal Molekülleri Adenovirüs aracılı Genetik Kaldırma

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embriyonik Fare Böbrek Nefrojenik Zone Hücreleri İzolasyon ve Kültür

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

Floresan Proteinleri Polarize Translokasyon Xenopus Wnt Sinyalizasyonu Cevabı Ektoderm

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

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. ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Kültürleme İnsan Embriyonik ve Bağlı Pluripotent Kök Hücrelerinin için uygundur Fare embriyonik fibroblast hücreleri hazırlanması

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Için bir sistem<em&gt; Ex vivo</emEmbriyonik Pankreas&gt; ekimi

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

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 ...