Bu protokol, Jane Dodd, Thomas Jessell ve Andrew Lumsden laboratuvarlarında geliştirildi. Kollajen omurilik kültürü eksplantlar Konrad Basler, Ann Calof Thomas Edlund, Phil Hamilton, Domna Karagogeos, Ariel Ruiz i Altaba ve Toshiya Yamada da dahil olmak üzere birçok kişi belirlendi. Marysia Placzek ve Marc Tessier-Lavigne akson rehberliği moleküllerinin belirlenmesi için bir araç olarak in vitro eksplantlar akson büyüme kullanarak tekniğinin öncülüğünü yaptı. Butler, laboratuvar İş NIH / NINDS Dimes ve R01 NS063999 Mart hibe tarafından desteklenmektedir.

Bölüm 1: transfekte COS Hücreler Agrega hazırlanması Asma Damlalar <ol><li> Tohum COS-7 (COS) 35mm kültür kaplarına hücreler. % 80 üretici s protokole göre Lipofectamine2000 kullanarak hücrelerin içine plazmid ifade confluency, transfect 1μg ulaştığınızda. </li><li> Damla asılı hazırlamak için, transfeksiyon orta aspirat ve transfekte COS hücreleri 1ml 1x Fosfat Tamponlu Çözümü (PBS) ile yıkayın. 0.5 ml, 15 dakika boyunca enzim ücretsiz cep disosiasyon orta hücreleri davranın. 1 ml reaksiyonu durdurmak için Opti-MEM + 1x Penisilin / Streptomisin / Glutamin (P / S / G) +% 10 fetal sığır serumu (FBS) bir çözüm ekleyin. </li><li> Karışım hücrelerin kültür çanak yüzeyinden bunları kaldırmak ve konik tüp 15ml içine aktarmak için. 2000K az 2 dakika boyunca pelet hücreleri Spin. Opti-MEM + 1x P / S / G +% 10 FBS 100μl süpernatant ve tekrar süspansiyon pelet çıkarın. </li><li> Invert çanak alt üst kapak ve yeri, 35mm kültür çanak kapağın iç tarafında birden fazla 20μl damla Spot ve hücrelerin toplam kadar birkaç saat için 37 ° C inkübatör bırakın. </li></ol> Bölüm 2: Dorsal Omurilik eksplantlar hazırlanması <ol><li> E11 sıçan embriyolar anne rahim inceleyin ve gerektiği kadar L15 orta buz üzerinde tutmak. </li><li> , Bilenmiş bir tungsten iğne ile ön ayakları tomurcuk hemen altındaki bölge, her embriyo gövde 4-6 segmentinde bir bölümü çıkarın. Plastik bir pipet kullanarak, 4-iyi Nunc çanak iyi bir doku parçaları toplamak ve buz üzerinde tutmak. </li><li> Tüm embriyo segmentleri disseke, 1ml L15 + Nunc çanak iyi bir ikinci 1mg dispase bir çözüm adet aktarmak için plastik pipet kullanın. 5 dakikadan daha uzun süre oda sıcaklığında inkübe edin. Dispase ile doku parçaları üzerinde inkübe etmeyin. </li><li> İnkübasyon sırasında, bir Petri kabı ve karıştırmak için girdap içine L15 hakkında 10ml ısı inaktive normal keçi serumu (HIGS) 0.5 ml ekleyin. Üçüncü bir Nunc çanak bu çözeltinin 1ml aktarın ve inkübasyon sona erdiğinde bu çözüm doku parçaları aktarmak. Buz üzerinde tutun. Not: doku parçaları, bir saat için buz üzerinde &quot;rest&quot; izin verilir sonraki diseksiyon daha kolay olacaktır. </li></ol> Bölüm 3: Astar Kollajen <ol><li> 360μl kollajen 40μl 10x Minimal Essential Orta ekle, tüp ya da kısaca vorteks Flicking hızlı ve iyice karıştırın. Bir picofuge hızla aşağı spin. Çözüm sarı olacaktır. Mümkün olduğu kadar buz üzerinde tutun. </li><li> (Aşağıdaki nota bakınız) biraz portakal kollajen çözüm açmak için yeterli 0.8M sodyum bikarbonat (NaHCO 3) ekleyin. Yine Flicking tarafından hızlı ve iyice karıştırın ve bir picofuge kısaca aşağı doğru döndürün. Çözüm karıştırıldıktan sonra pembe kalırsa, çok NaHCO 3 ekledik ve kollajen taze bir tüp ile tekrar başlatmak gerekecektir. </li><li> Bu noktada kollajen çözüm astarlanmalıdır. Buz sıvı kalır, ancak yaklaşık 5 dakika (pembe dönüm) oda sıcaklığına getirilmelidir zaman içinde daha da pekiştireceğiz. </li><li> 4-iyi Nunc çanak her iyi kollajen Spot 20μl. Pipetin ucu kullanarak, küçük bir &quot;pad&quot; oluşturmak için kolajen yayıldı. Oda sıcaklığında kollajen edelim. </li></ol> Not: NaHCO 3 tam miktarı, kolajen her parti için titre edilmesi gerekir . Düşük Başlangıç ​​(11μl) ve çözüm portakal hafif bir gölge kadar 0.5-1μl adım adım ekleyin. &quot;Doğru&quot; miktarı genellikle 1ul NaHCO 3 küçük miktarda kolajen pembe açacak daha az. NaHCO 3 miktarı doğrusal yukarı veya aşağı ölçekli değildir. Bölüm 4: Dorsal Omurilik eksplantlar ve Kollajen Matrix COS Hücre Agregalar Onları yerleştirilmesi Diseksiyon <ol><li> Taze bilenmiş tungsten, ince forseps bir iğne ve çift (# 5 veya # 55) kullanarak, omuriliği saran mezoderm çıkarın. </li><li> Tungsten iğne ile taban plakası ile paralel Kesme, omuriliğin ventral beşte dikkatlice çıkarın. </li><li> Kenarları mümkün olduğu kadar temiz kesilmiş olduğunu emin olun, dorsal spinal eksplant iki eşit parçaya böler. Gerekirse, kenarları kırpın. Yaklaşık çekti cam kapiller tüp ile donatılmış bir ağız pipet kullanarak ayrı kollajen pabuçlarına her dorsal spinal eksplant aktarın. Çapı 0.5mm. </li><li> Transfekte COS hücrelerinin toplam dorsal spinal eksplant lateral kenarı yaklaşık olarak aynı genişlikte kareler halinde kesin. </li><li> Ağız pipet kullanarak bu kareler, dorsal spinal eksplant ile kollajen ped üzerine aktarın. </li><li> Ağız pipet ile, ağız pipet kullanarak herhangi bir aşırı sıvı aspire. Aspirat bitmedi. </li><li> Ped üzerine boyanabilir kollajen 4μl uygulayın ve tungsten iğne kullanarakEksplantlar üzerinde kollajen doku doğrudan dokunmadan hareket ettirin. Eksplantlar çevreleyen kollajen tungsten iğne hareket ettirerek, şark eksplantlar COS hücrelerinin toplam dorsal spinal eksplant lateral kenarına bitişik olduğunu. </li><li> Kollajen sonra eksplantlar kollajen tamamen kaplı olduğundan emin olmak için kollajen 4μl uygulamayı tekrarlayın. </li><li> Bir doku kültürü kaputu, 30-40 saat Opti-MEM + 1xP/S/G ve kültür 0.5 ml ekleyin. </li><li> Eksplantlar, buz üzerinde plaka tutarak, 1xPBS olarak 0.5 ml% 4 paraformaldehid 45 dakika sabitleyin. </li><li> 1xPBS engelleyici bir çözüm +% 0.1 Triton-X100 +1% HIGS (PBTN) 0.5 ml ile iki kez yıkayın. Blok 4 ° C en az birkaç saat, bir gecelik inkübasyon immünohistokimyasal süreç, daha sonra tercih ve. </li></ol> Bölüm 5: İmmünohistokimya Commissural Aksonlar Görselleştirme <ol><li> PBTN ile bloke sonra, 48-iyi bir yemek yer ve forseps ile 4 plaka eksplantlar kesip. </li><li> Her iyi birincil antikor 200μl ekleyin ve 4 gece bekletin ° C. Commissural aksonlar görselleştirmek için, fare anti-tag1 antikor 01:06 çözümü kullanın. Transfeksiyon başarılı olup olmadığını belirlemek için, aynı zamanda sinyal molekülüne epitopu etiketli varsa ya sinyalleme molekülü test veya ilgili epitopu karşı primer antikor içerir. </li><li> 4 PBTN 5 x 1 saat ° C için her örnek yıkayın </li><li> Her iyi ikincil antikor 200μl ekleyin ve 4 gece bekletin ° C. Primer antikor olarak anti-tag1 antikorları kullanılmış olsaydı, ya FITC veya Cy3 birleştiğinde bir keçi anti-fare IgG sekonder antikoru kullanın. Işığa duyarlı ikincil antikorlar kullanılarak FITC veya Cy3 birleştiğinde, bu noktada folyo ile kaplanmış 48-iyi çanak tutmak. </li><li> Her numune yıkayın 5 ° C 4 x 1 saat PBTN içinde her </li><li> 3-iyi bir depresyon slayt eksplant transfer Vectashield orta ve kapak, bağlama, aşırı sıvı kaldırmak. 4 ° C veya -20 ° C ya da hafif bir kasa Mağaza </li></ol> Bölüm 6: eksplant Temsilcisi Görüntüler Eksplant ve COS agrega başarılı bir deneyde, birbirine bitişik kalır ve kollajen setleri dışında kayması değil. Aksonların minimal aşırı çoğalma olacak; önemli aksonal üremesine yol çok uzun süre dorsal spinal eksplant kültür olduğunu gösterir. Eksplant büyüyen hiçbir blep olacak; doku kültür ortamı ile doğrudan temas haline gelirse blebbing oluşur. Aksonların yeniden yönlendirmek için sinyal molekülü yeteneği konfokal mikroskopi kullanılarak değerlendirildi. Akson reorientation ölçüde COS hücre toplam 3 yakın aksonların yeniden yönlendirilmesi ortalama açı ölçümü ile belirlenebilir. Şekil 1&#39;de gösterildiği gibi, COS hücreleri ifade myc etiketli BMP7 (mavi) bir çatı plaka eksplant commissural aksonlar iter (Şekil şekilde benzer BMP7 kaynak (Şekil 1B) uzak tag1 + commissural aksonlar (yeşil) reorient 1A). Kontrol vektör ifade COS hücreleri commissural aksonlar 3,4 yönünü üzerinde hiçbir etkisi yoktur. Bu eksplantlarında da motor nöronlar ve dorsal internöronlar süsleyen transkripsiyon faktörü Isl1 / 2 (kırmızı) karşı antikorlar ile işaretlenmiştir. Bu sonuç, ilk belirtisi olduğunu BMP aile aracılık üyesi çatı plakası chemorepellent 3 aktivitesi. <img src="/files/ftp_upload/1853/1853fig1tmb.jpg" border="0" /> Şekil 1: Lütfen Şekil 1&#39;de bir büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1853/1853fig1.jpg">buraya tıklayınız</a> .

<ol>
	<li>Altman, J., Bayer, S. 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> The development of the rat spinal cord. , (1984).</li><li>Placzek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+recombinations+in+collagen+gels.">Tissue recombinations in collagen gels.</a> Methods in molecular biology. , 325-335 (2008).</li><li>Augsburger, A., Schuchardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPs+as+mediators+of+roof+plate+repulsion+of+commissural+neurons.">BMPs as mediators of roof plate repulsion of commissural neurons.</a> Neuron. 24, 127-141 (1999).</li><li>Butler, S. J., Dodd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+BMP+heterodimers+in+roof+plate-mediated+repulsion+of+commissural+axons.">A role for BMP heterodimers in roof plate-mediated repulsion of commissural axons.</a> Neuron. 38, 389-401 (2003).</li><li>Kennedy, T. E., Serafini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins+are+diffusible+chemotropic+factors+for+commissural+axons+in+the+embryonic+spinal+cord.">Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.</a> Cell. 78, 425-435 (1994).</li><li>Charron, F., Stein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphogen+sonic+hedgehog+is+an+axonal+chemoattractant+that+collaborates+with+netrin-1+in+midline+axon+guidance.">The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance.</a> Cell. 113, 11-23 (2003).</li><li>Lumsden, A. G., Davies, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotropic+effect+of+specific+target+epithelium+in+the+developing+mammalian+nervous+system.">Chemotropic effect of specific target epithelium in the developing mammalian nervous system.</a> Nature. 323, 538-539 (1986).</li><li>Dodd, J., Morton, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+regulation+of+axonal+glycoprotein+expression+on+subsets+of+embryonic+spinal+neurons.">Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons.</a> Neuron. 1, 105-116 (1988).</li><li>Yamauchi, K., Phan, K. D., Butler, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+type+I+receptor+complexes+have+distinct+activities+mediating+cell+fate+and+axon+guidance+decisions.">BMP type I receptor complexes have distinct activities mediating cell fate and axon guidance decisions.</a> Development. 135, 1119-1128 (2008).</li></ol>

Bu testte gerçekleştirme başarısını belirlemek kritik faktörler, doku, çok uzun süre bir süre için dispase ile tedavi edilmesi gerektiğini, bu tedavi çok yapışkan hale doku ve canlılığı azalmış olan yol açacaktır. İki, kollajen mükemmel astar ve mümkün olduğu kadar buz üzerinde muhafaza edilmelidir. Ayarlamaya başlıyor işlemek için giderek daha zor olacak, yani pembe. Üç, tungsten iğne her zaman çok keskin tutulması gerekir.

assaying the ability of diffusible signaling molecules to reorient embryonic spinal commissural axons

Omurgalı omurilik 1 Dorsal commissural aksonlar aksona yol sinyalleri tanımlamak için paha biçilmez bir model sistem var. Burada, in vitro testi commissural aksonlar yönünü 2 dışsal ve içsel sinyalleri etkisini araştırmak için yaygın olarak kullanılan &quot;yeniden yönlendirilmesi assay&quot; açıklar. Bu testte, BMP, chemorepellent dahil olmak üzere, çok sayıda kişi tarafından Jane Dodd, Thomas Jessell ve Andrew Lumsden laboratuarlarında geliştirilen (Daha fazla bilgi için teşekkürler) ve bu testte sürümlerini anahtar akson rehberlik molekülleri yeniden yönlendirilmesi faaliyetleri göstermek için kullanılan 3,4 ve Netrin1 5 ve Sonic Hedgehog (Shh) omurilik zemin plakasını 6 chemoattractive faaliyetleri çatı plakası. Eksplantlarındaki 2-3 segmentlerinde omurilik dorsal üçte ikisini oluşturan embriyonik gün (E) 11 sıçan disseke ve üç boyutlu kolajen jeller 7 olarak kültür. E11 dorsal spinal eksplantlar glikoprotein, tag1 8 aksonal ifade tarafından tespit edilebilir, yeni doğan commissural nöronlar, içerir . Commissural akson yörünge kültür 30-40 saat boyunca, in vivo olarak görülen benzer bir ders ile bu sırt eksplantlar değinmeyecek. Bu aksonal yörünge test dokuları ya da dorsal eksplant lateral kenarlarından biri ile temas aday sinyalleme molekülü ifade eden bir COS hücre agrega ya koyarak itiraz edilebilir. Eklenen doku çevresinde uzanan Commissural aksonlar endojen çatı plaka ve ektopik lateral doku sinyalleri hem de etkisi altında büyüyecek. Bu koşullar altında commissural aksonlar Nokia&#39;nın derecesi belirlenebilir. Bu testte kullanarak, hem de belirli bir sinyal commissural yörünge 9 doğrudan bu sinyal gerekliliği sıra commissural aksonlar 3,4 reorient yeterliliğini incelemek mümkündür.

Watch this Scientific Journal Video about Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons at JoVE.com

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Bu testte commissural aksonların yeniden yönlendirmek için bir sinyal molekülü yeteneği, burada kemik morfogenetik protein 7 (BMP7) değerlendirir. Embriyonik dorsal spinal kord bir eksplant COS hücreleri salgılayan aday büyüme faktörleri toplu bir komşu kültür. Eksplant içinde büyüyen Nokia&#39;nın commissural aksonlar immünohistokimya ile görüntülendi.

Embriyonik Spinal Commissural Aksonlar yönünü difüze Sinyal Molekülleri yeteneği Assaying

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

assaying-ability-diffusible-signaling-molecules-to-reorient-embryonic

Research

JoVE Journal

Biology

8.8K Görüntüleme.  University of Southern California. Bu testte commissural aksonların yeniden yönlendirmek için bir sinyal molekülü yeteneği, burada kemik morfogenetik protein 7 (BMP7) değerlendirir. Embriyonik dorsal spinal kord bir eksplant COS hücreleri salgılayan aday büyüme faktörleri toplu bir komşu kültür. Eksplant içinde büyüyen Nokia'nın commissural aksonlar immünohistokimya ile görüntülendi.

Video: Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Nöron Soma ve Aksonlar compartmentalization mikroakışkan Aygıt Fabrikasyon

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Biyoloji

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Omurilik Elektrofizyoloji

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Fare Embriyonik Akciğer Kültür, Dallanma Moleküler Mekanizmalar değerlendirin Bir Sistem

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

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 ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Embriyonik Kök Hücreler Yaşayan Kromatin Protein Dynamics Tedbir Tahliller (sıkı bağlamak ve FLIP) Photobleaching

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

LRRK2 Kinaz Aktivitesi Assaying In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

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 ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Blastomer Eksplantlarından Embriyonik gelişim sırasında Hücre Kader Taahhüt Test Etmek için

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Hücre Tek katmanlar mekanik uyarılması bağlı Kalsiyum Dalga Yayılım: Sığır Kornea Endotel Hücreleri Örneği

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).