Yazarlar, Toronto, Ontario bölgesindeki Mount Sinai Hastanesi Dr. Rizaldy Scott birincil Rac1 Flox / Flox fare embriyonik fibroblastlar onun hediye için teşekkür etmek istiyorum . Bu çalışma, Kanada Sağlık Araştırmaları (CIHR, 2.009-93.526 MOP) ve Kanada Doğa Bilimleri ve Mühendislik Araştırma Konseyi (NSERC 327.372-06 RG) Enstitüleri NJ hibe işletim tarafından desteklenmiştir. SH ve MW CGSMA ve sırasıyla CIHR ve NSERC, KKS-M ödül tarafından desteklenmektedir. Steve Hawley ve Melanie Wills bu kağıt için aynı derecede katkıda bulunmuştur.

Çözülme hücreleri <ol><li> Sıvı azot donmuş fare embriyonik fibroblastlar (MEFS) (bireysel embriyo primer kültürlerden gelen, daha önce oluşturulan) alın. </li><li> 37 flakon çeker çözülme hücreleri ° C su içeriğini tamamen çözülmüş kadar dönen. </li><li> 10cm hücre kültürü plakası% 10 fetal sığır serumu ve% 1 penisilin-streptomisin ile takviye edilmiştir DMEM 9ml ekleyin. </li><li> Plaka açılan akıllıca çözülmüş hücre süspansiyonu ekleyin, 37 ° C,% 5 CO 2 inkübatör gecede hücrelerin ve dağıtmak için yer plaka plaka hafifçe sallayın. Hücreleri DMSO (donma süreci) duyarlı hücreleri (yaklaşık dört saat sonra kaplama) plaka yapıştırılır sonra, ilk medya taze medya ile değiştirilmesi olduğunu unutmayın. Ayrıca çözülme sonrasında bu hücrelerin izdiham dikkat donmuş hücreleri sayısı bağlıdır, hücrelerin dondurulması sonra iyileşme oranı ve hücrelerin büyüme oranı. </li></ol> Pasajlanması hücreleri <ol start="5"><li> Hücreleri confluency aslında% 100 (yani: ertesi gün) büyüdü, medya aspirat steril PBS 5ml ile hücre yıkayın. </li><li> PBS çıkarın ve yaklaşık 5 dakika için inkübatör 10mM EDTA yeri ve plakası içeren 1 ml tripsin eklemek, hücreleri plaka ayırmak için izin verecek. </li><li> Hücreleri müstakil, tripsinize hücreleri kümeleri dağıtmak, medya 9ml içeren yeni bir plaka üzerine açılan bilge bu süspansiyon 1mL eklemek, ve bu yerde 37 ° C inkübatör gecede medya 9ml eklemek, pipet 2-3 kez . Alternatif medya FBS varlığı tripsin inaktive, hücreler tripsin sulandırmak için ilk yıkanmış olabilir. </li></ol> Kapak fişleri üzerine hücrelerinin sayımı ve kaplama <ol start="8"><li> Inkübatör gelen hücreleri çıkarmak, bu sefer yerine 9ml tripsinize hücreleri 5-6mL ortam eklemek dışında, yıkama ve daha önce olduğu gibi trypsinize. </li><li> Pipet hücreleri tek bir hücre süspansiyonu iyice içine tam ayrılma sağlamak ve ayrı bir mikrofuge&#39;de boru tripan mavi 15μL ile birleştirmek için 15μL kaldırmak. </li><li> Bu hücre / tripan mavisi süspansiyon birkaç kez aşağı yukarı pipetleme Mix ve hemasitometre 15μL ekleyin. </li><li> Mikroskop altında, ölü hücreleri mavi görünecektir. Hemasitometre 4X4 ızgaraları birinde renksiz / clear (yaşam) hücrelerinin sayısını. Diğer üç 4X4 ızgaraları (gösterilir A, B, C ve D) Bu sayımı tekrarlayın ve süspansiyon mcL başına hücre sayısını hesaplamak için aşağıdaki denklemi kullanın: <img src="/files/ftp_upload/2160/2160eq0.jpg" alt="Denklem 1" /></li><li> Protokol optimizasyonu sayesinde, deney sonuca görünüm için uygun hücre yoğunluğu 30.000 hücreleri sonuçları kaplama olduğunu tespit ettik. </li><li> 30.000 hücreleri için gerekli olan hücre süspansiyonu hacmini hesaplamak için, aşağıdaki denklemi kullanın: <img src="/files/ftp_upload/2160/2160eq1.jpg" alt="Denklem 2" /></li><li> Şimdi altı sıra çanak her bir kuyunun içine steril bir cam kapak kayma yeri ve her iyi medya 2mL eklemek ve ardından hücre süspansiyonu hacmi 30.000 hücreleri için gerekli olan her bir kuyunun içine açılan bilge eklemek. Biz daha sonraki bir zaman noktasında floresan mikroskobu kullanarak hücrelerin görselleştirme olacak gibi, bu deney için kullanılan cam kapak fişleri olduğunu unutmayın; tüm uygulamalar mutlaka mikroskopi gerektirir ve böylece fişleri gerekli olmayabilir kapsayacak. </li></ol> Virüs İletimi <ol start="15"><li> Birkaç saat veya gece boyunca (hücrelerin kapak fişleri uymasını sağlamak için yeterli bir süre) sonra, medyayı çıkartın ve 1 ml serum serbest DMEM hücreleri yıkayın. </li><li> Ayrıca virüs için her bir kuyu için 1 ml serum ücretsiz medya ekleyin. Bu deneme için, adeno-Cre virüs vektör Biolabs ticari olarak elde edilmiştir. </li><li> Önceki protokol optimizasyonu 500 enfeksiyon çokluğu (İçişleri Bakanlığı) birincil MEFS hücre canlılığı çok az veya hiç etkisi ile yüksek verimli gen transdüksiyonu sağladığını göstermiştir. İçişleri Bakanlığı aşağıdaki gibi hesaplanacaktır: <img src="/files/ftp_upload/2160/2160eq2.jpg" alt="Denklem 3" /> <img src="/files/ftp_upload/2160/2160eq3.jpg" alt="Denklem 4" /></li><li> Enfekte olduğu her iyi virüs doğrudan hesaplanan hacim ekleme, plaka gecede 37 ° C inkübatör içine virüs ve yer hücrelerini dağıtmak için hafifçe sallayın. </li><li> Ertesi sabah, kaldırmak için virüs içeren hücreler medya ve 1ml steril PBS içinde hücre yıkayın ve sonra her bir kuyu için düzenli medya 2mL eklemek. </li><li> Bu deneyde örneklediği Rac1 hücrelerinin durumda, yaklaşık 72 saat sonrası transdüksiyon hücreleri morfolojik değişiklikler meydana geldi. </li></ol> Temsilcisi Sonuçlar Hücreler görselleştirmek edildid aktin boya Phalloidin (sadece görüntüleme amaçları için) boyama ve Guelph Üniversitesi İleri Analiz Merkezi Leica TCS-SP5 multiphoton konfokal lazer tarama mikroskobu kullanılarak görüntülü. Şekil 1, virüsü değil, aynı hücrelere göre adeno-Cre virüse maruz Rac1 Flox / Flox hücreleri sözleşmeli ve uzamış morfolojisi gösterir . <img src="/files/ftp_upload/2160/2160fig1.jpg" alt="Şekil 1" /> Şekil 1 (sağda) aynı kaynaktan enfekte olmamış hücrelerdeki göre adeno-Cre virüsü (solda) ile enfekte Rac1 Flox / Flox hücreleri arasındaki karşılaştırma.

<ol>
	<li>Guo, F., Debidda, M., Yang, L., Williams, D. A., Zheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Deletion+of+Rac1+GTPase+Reveals+Its+Critical+Role+in+Actin+Stress+Fiber+Formation+and+Focal+Adhesion+Complex+Assembly.">Genetic Deletion of Rac1 GTPase Reveals Its Critical Role in Actin Stress Fiber Formation and Focal Adhesion Complex Assembly.</a> J. Biol. Chem. 281, 18652-18659 (2006).</li><li>Glogauer, M., Marchal, C. C., Zhu, F., Worku, A., Clausen, B. E., Foerster, I., Marks, P., GP, D. o. w. n. e. y., Dinauer, M., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1+deletion+in+mouse+neutrophils+has+selective+effects+on+neutrophil+functions.">Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions.</a> J. Immunol. 170, 5652-5657 (2003).</li><li>Prost, S., Sheahan, S., Rannie, D., Harrison, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+Cre+deletion+of+floxed+sequences+in+primary+mouse+cells+is+an+efficient+alternative+for+studies+of+gene+deletion.">Adenovirus-mediated Cre deletion of floxed sequences in primary mouse cells is an efficient alternative for studies of gene deletion.</a> Nucleic Acids Res. 29, E80-E80 (2001).</li><li>Rijnkels, M., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-Cre-mediated+recombination+in+mammary+epithelial+early+progenitor+cells.">Adenovirus-Cre-mediated recombination in mammary epithelial early progenitor cells.</a> J. Cell. Sci. 114, 3147-3153 (2001).</li><li>Vidali, L., Chen, F., Cicchetti, G., Ohta, Y., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1-null+mouse+embryonic+fibroblasts+are+motile+and+respond+to+platelet-derived+growth+factor.">Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor.</a> Mol. Biol. Cell. 17, 2377-2390 (2006).</li><li>Wang, H., Xie, H., Zhang, H., Das, S. K., Dey, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+recombination+by+adenovirus-driven+Cre+in+the+mouse+uterus.">Conditional gene recombination by adenovirus-driven Cre in the mouse uterus.</a> Genesis. 44, 51-56 (2006).</li></ol>

Hayvan Bakım Kanada Konseyi tarafından belirlenen kurallara ve yönetmeliklere uygun olarak hayvanlar üzerinde deneyler yapıldı.

İlişikteki video rekombinant virüs Cre recombinase kullanarak in vitro tüketim belirli bir gen nasıl kullanılabileceğini örnek teşkil etmektedir. Ancak bu protokolün geliştirilmesi ele değer bazı belirli noktaları ortaya yaptı. <ul><li> Adenovirüs ile çalışırken uygun güvenlik prosedürleri, her zaman kullanılmalıdır. Virüs ile çalışırken her zaman bir laboratuvar önlüğü ve eldiven giyilmelidir. Medya ve plastik eşya, en az 30 dakika süreyle% 10 oranında çamaşır suyu ile tedavi edilmelidir ve plastik eşya bundan sonra otoklava olmalıdır. </li><li> Kaçının tekrarlanan donma-çözülme, bu virüs titresi etkileyebilir ve böylece transdüksiyon etkinliğini azaltmak gibi virüs stokunun. Küçük miktarda virüs stokunun başlangıçta ayrı tüpler içine aliquoted olmalıdır. </li><li> 50-500 gelen değişen Mois yöntemi geliştirme sırasında test edildi ve verimli transdüksiyon oranları hücre canlılığı üzerine olumsuz etkileri olmadan, çoğu durumda gözlendi. </li><li> Adeno-GFP transdüksiyon verimlilikleri hızla ekrana yöntem geliştirme sırasında kullanıldı. Bu kontrol (veya boş bir vektör), hücresel değişikliklere neden olmaz sadece bu viral enfeksiyon sağlamak için deney sırasında yapılmalıdır. Aynı şekilde, hücrelerde ilgi floxed gen eksize herhangi bir hücresel değişiklikler (yani morfoloji veya canlılığı değişiklikler) neden değilse, o zaman adeno-GFP ile aynı hücre tipi bulaşmasını virüs transdüksiyon göstermek için kullanmak önemli bir kontrol meydana gelen. </li></ul> Kültür kaskadlar sinyalizasyon inceleme ötesinde, adeno-Cre yaklaşımı da, deney hayvanları 3,4 genler koşullu kaldırın ve 6 içinde bir organizmanın hücreleri belirli toplumlarda etiket in vivo olarak istihdam edilmiştir . Konak hücre içine Cre recombinase başka genlerin de tanıtmak için adenovirüs sistemi adapte edilebilir. Bu teslim vektör kullanarak iki temel avantajı, yüksek iletimi ve gen teslim verimliliği ve konak DNA ile entegrasyon eksikliği. Ancak, yeni rekombinant adenovirüs nesil Emek yoğun olabilir. Burada açıklanan protokol bu nedenle daha önce geliştirilen adeno-Cre virüs istismar adenoviral sistemi gücünü sürdürür ve kaplin faiz, Rac1 floxed allel. Bu deney koşullu vuruş sayesinde, MEFS floxed Rac1 allel taşıyan adeno-Cre virüs kullanarak transdüksiyon gerçekten Rac1 eksikliği hücreleri 2,5 hücresel morfoloji karakteristik değişiklikleri önde gelen Rac1 eksizyonu neden olduğunu doğrulamıştır.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ad-CMV-Cre virus</td>
<td>&nbsp;</td>
<td>Vector Biolabs</td>
<td>1045</td>
<td>&nbsp;</td>
<tr>
<td>DME High glucose media 500mL</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SH3002201</td>
<td>&nbsp;</td>
<tr>
<td>FBS Canadian origin 500mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CAA15-701</td>
<td>&nbsp;</td>
<tr>
<td>PEN-STREP 1X solution 100mL</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SV30010</td>
<td>&nbsp;</td>
<tr>
<td>Texas Red-X phalloidin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>T7471</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T4549</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

adenovirus-mediated genetic removal of signaling molecules in cultured primary mouse embryonic fibroblasts

Genetik olarak çeşitli hücre sinyalizasyon kaskadlar belirli bileşenleri kaldırmak için modern sinyal iletimi analizi yeteneği ayrılmaz bir araç olmuştur. Cre-loxP sisteminin kullanımı yoluyla bu koşullu silme işlemini gerçekleştirmek için özel bir yöntem. Bu yöntem ile ilgi gen Cre recombinase protein için özel tanıma dizileri loxP siteleri, yan içerir. Sözde floxed (loxP sitesi ile çevrili) DNA Cre-aracılı bir rekombinasyon loxP siteleri olay ve müdahale gen 3 sonraki eksizyonu bu enzim sonuçlarına maruz kalması. Cre recombinase ilgi site yönetmek için birkaç farklı yöntem bulunmaktadır. Bu video, floxed Rac1 allel 1 içeren embriyolar elde edilen birincil fare embriyonik fibroblastlar (MEFS) bulaştırmak için Cre recombinase gen içeren bir adenovirüs kullanımını göstermektedir. Deneyler için Rac1 MEFS seçimi için gerekçe bu aktin hücre iskeletinin 2,5 değişiklikler nedeniyle Rac1 silinmesi, üzerine belirgin morfolojik değişiklikler görülebilmektedir. Viral iletimi ve Cre ifade takiben 72 saat, hücreleri aktin boya Phalloidin kullanılarak boyandı ve konfokal lazer tarama mikroskobu kullanılarak görüntülü edildi. Adeno-Cre virüsüne maruz kalmış MEFS, enfekte olmamış hücrelere oranla daha önceki raporlarda 2,5 tutarlı sözleşmeli ve morfolojisinde uzatılmış ortaya çıktığını gözlendi. Cre recombinase teslim adenovirüs yöntem adeno-Cre virüs kolaylıkla ulaşılabilir olduğu gibi avantajlı ve hücrelerin yaklaşık% 100 Cre yoluyla gen delesyonu optimize edilmiş adenoviral enfeksiyonu ile elde edilebilir.

Watch this Scientific Journal Video about Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts at JoVE.com

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

Bu video floxed Rac1 allel taşıyan birincil fare embriyonik fibroblastlar bulaştırmak için Cre recombinase gen taşıyan bir adenovirüs kullanın.

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 ...

adenovirus-mediated-genetic-removal-signaling-molecules-cultured

Research

JoVE Journal

Biology

24.8K Görüntüleme.  University of Guelph. Bu video floxed Rac1 allel taşıyan birincil fare embriyonik fibroblastlar bulaştırmak için Cre recombinase gen taşıyan bir adenovirüs kullanın.

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

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

İlköğretim Kültür Hipokampal Nöronlar Yoğun çekirdekli veziküller Canlı Görüntüleme

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Biyoloji

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Dört Transkripsiyon Faktör Fare Embriyonik Fibroblastlar yeniden programlanması Bağlı Pluripotent Kök Hücre Üretimi, Doksisiklin lentiviral İletim Sistemi Đndüklenebilir

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

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

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons2. This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate3,4 and the chemoattractive activities of Netrin15 and Sonic Hedgehog (Shh)6 in the floor plate in the spinal cord.
Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels7. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag18. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons3,4 as well the necessity for this signal to direct the commissural trajectory9.

This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.

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 ...

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 ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Yüksek Yoğunluklu İnsan Embriyonik Kök Hücre Kültürleri gelen Rapid Fibroblast Kaldırma

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

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 ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Saç Rejenerasyon sırasında epitelyal-mezenkimal Sinyal Rapid Genetik Analizi

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

Kültürlü organel Ulaşım<em&gt; Drosophila</em&gt; Hücreler: S2 Hücre Hattı ve İlköğretim Nöronlar.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Glikoz Alımı Ölçümü ve İnsülin Uyarıcısına Yanıt<em&gt; In vitro</em&gt; Kültürlü İnsan Primer Miyotları

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

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

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

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 ...