وقد تم تمويل هذا العمل من قبل DE018234 - R01 R01 وDE019638.

1. إعداد الأنسجة المانحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تعد وسائل الإعلام والثقافة ، وصقل الزجاج الدبابيس ، وشحذ الإبر التنغستن. </li><li style=";text-align:right;direction:rtl"> جمع الجنين من قذيفة ، ويغسل في الجليد الباردة برنامج تلفزيوني. <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> باستخدام الحقنة 10 مل و إبرة قياس 18 مل من 1.0 إزالة الزلال من نهاية وأشار من قشرة البيضة. </li><li style=";text-align:right;direction:rtl"> جعل ثقب صغير في الجزء العلوي من الغلاف باستخدام مقص نقطة ، وقطع دائرية ثم فتح للكشف عن الجنين </li></ol></li><li style=";text-align:right;direction:rtl"> إزالة الرأس والمكان الى DMEM (المصل الحرة ، ودرجة حرارة الغرفة) </li><li style=";text-align:right;direction:rtl"> عملية تشريح الجبهية من الجنين (أو غيرها من الجهات المانحة مثل موقع عملية الفك العلوي أو الفك السفلي) </li><li style=";text-align:right;direction:rtl"> في هضم 2.4U / مل dispase في برنامج تلفزيوني على الجليد ل20mins </li><li style=";text-align:right;direction:rtl"> تغطية الأنسجة مع DMEM مع جيش صرب البوسنة 1 ٪ لوقف عملية الهضم </li><li style=";text-align:right;direction:rtl"> استخدام الإبر التنغستن شحذ إلى الأديم الظاهر منفصلة ، اللحمة المتوسطة ، وneuroectoderm </li><li style=";text-align:right;direction:rtl"> تخزين الأنسجة الكسب غير المشروع في جيش صرب البوسنة DMEM 1 ٪ على الجليد حتى المضيفة مستعدة للزرع </li></ol> 2. إعداد المضيف <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تعرض الجنين <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> باستخدام الحقنة 10 مل و إبرة قياس 18 مل من 1.0 إزالة الزلال من نهاية وأشار من قشرة البيضة. </li><li style=";text-align:right;direction:rtl"> جعل ثقب صغير في الجزء العلوي من الغلاف باستخدام مقص نقطة. ضع قطعة من الشريط فوق الحفرة ، وقطع دائرية ثم فتح للكشف عن الجنين. </li></ol></li><li style=";text-align:right;direction:rtl"> 2 به زوجا من الملقط ، فهم السلى وبلطف المسيل للدموع لإحداث الثقب. </li><li style=";text-align:right;direction:rtl"> استدارة الرأس بوضع زوج من ملقط على العين اليمنى والضغط. في حين عقد رئيس مطرد ، واستخدام إبرة لإزالة التنغستن الأديم الظاهر المضيف لاستيعاب الكسب غير المشروع. </li><li style=";text-align:right;direction:rtl"> نقل الكسب غير المشروع إلى المضيف باستخدام ماصة الزجاج. </li><li style=";text-align:right;direction:rtl"> موقف الكسب غير المشروع لتحل محل الأديم الظاهر إزالتها. اضافة الى وجود دبوس الزجاج في كل زاوية من النسيج المطعمة يعلقون الكسب غير المشروع في المكان. دبابيس لجعل الزجاج ، وسحب أنبوب microcapillary إلى نقطة غرامة على مدى اللهب الكحول. </li><li style=";text-align:right;direction:rtl"> مكان الشريط بإحكام خلال ثقب والعودة الجنين الى حاضنة لمدة مناسبة من الوقت لتحليلها. </li><li style=";text-align:right;direction:rtl"> انظر أيضا : 9 </li></ol> 3. ممثل النتائج : لتقييم الوهم ، ينبغي جمع الأجنة ومعالجتها لتحليل توزيع المضيفة والمانحة الأنسجة. للكشف عن خلايا السمان ، المناعية باستخدام أجسام مضادة QcPN (الموصوفة في : 3) يعمل ، وللكشف عن خلايا فأر في التهجين الموقع على أقسام البارافين يستخدم 6. وينبغي أن يقتصر الخلايا المانحة للظهارة ، وينبغي أن يكون هناك أي دليل على أنسجة المانحة الوسيطة (الشكل 1). وجود خلايا المانحة المعزولة في اللحمة المتوسطة يدل على وجود الخلايا العصبية التي تلوث قمة سوف نخلط أي تفسير الشكلية نظرا لتأثير هذه الخلايا على العديد من جوانب التنمية الوجه (على سبيل المثال ، 10). مرة واحدة على اقتناع بأن تقنية خالية من الكسب غير المشروع هو تلويث اللحمة المتوسطة ، ويمكن استخدام مزيد من التدابير نتائج المورفولوجية أو الجزيئي لتقييم الوهم. لأغراضنا ، اكتشفنا وجود حمل خارج الرحم الغضاريف والعظام التي تتوافق مع الازدواجية في الفك العلوي التي يسببها الأنسجة المزروعة (الشكل 2) ، والتغيرات الجزيئية في الأنسجة الوسيطة ردا على المطعمة الأديم الظاهر (الشكل 3) 5،6. <img src="/files/ftp_upload/2557/2557fig1.jpg" alt="الشكل 1" /> الشكل 1. تقييم الخيمرية يستخدم (أ) مكافحة QcPN تلوين للكشف عن خلايا في السمان السمان ، فرخ الوهم. لا يوجد أي دليل على تلطيخ في اللحمة المتوسطة. يستخدم (B) في الموقع التهجين للكشف عن التعبير النصوص B2 شرط في الماوس الفرخ الوهم. ويقتصر التعبير على الأديم الظاهر تضم الكسب غير المشروع. <img src="/files/ftp_upload/2557/2557fig2.jpg" alt="الشكل 2" /> الشكل 2. تلوين ثلاثي الألوان يظهر ازدواجية هيكل عظمي الفك العلوي في خيمر السمان ، الفرخ (A) و (B) الوهم الماوس الفرخ. <img src="/files/ftp_upload/2557/2557fig3.jpg" alt="الشكل 3" /> الشكل 3 ، والأديم الظاهر regultes التعبير الجيني في اللحمة المتوسطة. (أ) عادي Bmp7 التعبير في اللحمة المتوسطة من جنين الفرخ (المشعة في الموقع التهجين مع الإيجابية الزائفة أحمر اللون في إشارة أدوبي فوتوشوب). (ب) هي التي يسببها Bmp7 التعبير في اللحمة المتوسطة المتاخمة للطعم (السهم) في أجنة خيالية.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+can+form+cartilages+normally+derived+from+mesoderm+during+development+of+the+avian+head+skeleton.">Neural crest can form cartilages normally derived from mesoderm during development of the avian head skeleton.</a> Developmental Biology. 208, 441-441 (1999).</li><li>Couly, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+Hox-negative+cephalic+neural+crest+cells+and+the+foregut+endoderm+in+patterning+the+facial+skeleton+in+the+vertebrate+head.">Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head.</a> Development. 129, 1061-1061 (2002).</li><li>Evans, D. J., Noden, D. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+repetitive+mouse+B2+element+to+identify+transplanted+mouse+cells+in+mouse-chick+chimeras.">Use of a repetitive mouse B2 element to identify transplanted mouse cells in mouse-chick chimeras.</a> Exp Cell Res. 248, 75-75 (1999).</li><li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+chicken+eggs+for+developmental+studies.">Windowing chicken eggs for developmental studies.</a> J Vis Exp. , (2007).</li><li>Eames, B. F., Schneider, R. 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الإعلان عن أي تضارب في المصالح.

باستخدام هذا الأسلوب زرع سمح لنا لتحديد الأديم الظاهر أن يحتوي على معلومات يشير إلى أن ينظم قطبية ظهري بطني والإرشاد proximodistal من الفك العلوي. التشابه في النتائج عند استخدام الماوس السمان أو الأديم الظاهر ، والحفاظ على الاشارات الجزيئية في هذا النسيج من بين العديد من الأنواع 6،11 يشير إلى أن هذا هو مركز للغاية الحفظ إشارات بين الفقاريات. وعلاوة على ذلك ، استخدمت محققين آخرين تقنيات مشابهة لاختبار خصائص إشارات من مناطق مختلفة من الأديم الظاهر سطح رأسي وكشفت وجود العديد من المراكز التي تقع في إشارة الأديم الظاهر التي تسهم في التشكل في الوجه 12. هذا الأسلوب يتيح لنا لزيادة فهمنا للدور الذي الأنسجة المختلفة ، فضلا عن الاختلافات الإقليمية داخل الأنسجة ، ولعب خلال التنمية الوجه. أخيرا ، من خلال الجمع بين قوة الوراثة الفأر مع قوة النظام الطيور وخيمر سوف نكون قادرين على إلقاء الضوء على وسطاء من التفاعلات الجزيئية ، الظهارية الوسيطة خلال التنمية ، وسوف نكون قادرين على تحديد الآليات التي تنظم هذه التفاعلات التنمية.

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assessing signaling properties of ectodermal epithelia during craniofacial development

وقد ساعدت سهولة الحصول على أجنة الطيور علم الجنين التجريبي فهم مصائر الخلايا أثناء التطور ودور الأنسجة التي تنظم التفاعلات الزخرفة والتشكل من الفقاريات (على سبيل المثال ، 1 ، 2 ، 3 ، 4). هنا ، نحن لتوضيح أسلوب الذي يستغل هذا الوصول لاختبار الإشارات وخصائص الزخرفة الأدمة من أنسجة الوجه أثناء تطوير. في هذه التجارب ، فإننا نخلق السمان ، فرخ 5 أو الماوس الفرخ 6 من زرع الوهم في الأديم الظاهر السطحي الذي يغطي الرأس والفك العلوي من السمان أو الماوس على المنطقة إما نفسه أو في منطقة خارج الرحم لافراخ الدجاج. وقد تم تطوير استخدام السمان والأنسجة المانحة للزرع في الدجاج للاستفادة من علامة موجودة في نويي السمان ولكن الخلايا لا الفرخ ، وبالتالي السماح للمحققين تميز المضيفة والمانحة الأنسجة 7. وبالمثل ، وهو عنصر المتكررة موجودة في جينوم الفأر ، ويعبر بتواجد مطلق ، والذي يسمح لنا أن نميز بين المضيفة والمانحة في الأنسجة الفرخ الفأر الوهم 8. سيتم استخدام الماوس كما الأديم الظاهر تمديد الأنسجة المانحة إلى حد كبير فهمنا لهذه التفاعلات الأنسجة ، وذلك لأن هذا سوف يسمح لنا لاختبار خصائص الإشارات من الأديم الظاهر المستمدة من أجنة متحولة مختلفة.

Watch this Scientific Journal Video about Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development at JoVE.com

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

توضح هذه المقالة تقنية زرع الانسجة التي تم تصميمها لاختبار الإشارات وخصائص الزخرفة من الأديم الظاهر سطح الرأس أثناء تطوير القحفي.

تقييم خصائص اشارة من الظهارة الأدمة خلال التنمية الجمجمة

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

assessing-signaling-properties-ectodermal-epithelia-during

Nanyang Technological University

Research

JoVE Journal

Biology

9.4K Views.  University of California San Francisco. توضح هذه المقالة تقنية زرع الانسجة التي تم تصميمها لاختبار الإشارات وخصائص الزخرفة من الأديم الظاهر سطح الرأس أثناء تطوير القحفي.

Video: Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

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.

Electroporation من اللحمة المتوسطة الجمجمة

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

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.

تحليل سريع لالوراثية طلائي-الوسيطة اشارة خلال تجديد الشعر

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

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

تتبع الخلية عن طريق البروتينات Photoconvertible خلال التنمية اسماك الزرد

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

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.

إإكسبلنتس قسيم أرومي لاختبار الالتزام مصير الخلايا أثناء التطور الجنيني

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.

تحفيز الميكانيكية التي يسببها الكالسيوم انتشار الموجات في الخلية monolayers: مثال من الأبقار الخلايا البطانية القرنية

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

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

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

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

التصور التنمية الجمجمة في sox10: Kaede الذي الزرد المعدلة وراثيا عن طريق الخط الوقت الفاصل بين متحد البؤر المجهري

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

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

الأيضية وصفها وغشاء التقطير للالمقارن تحليل البروتين من<em&gt; نبات الأرابيدوبسيس thaliana</em&gt; الثقافات تعليق خلية

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

أسلوب الثقافة شريحة لوبعد تطور الجراثيم الأسنان في يزدرع الثقافة

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

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

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

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

الثقافات البديلة لافرة القوة البشرية الجذعية إنتاج الخلية، الصيانة، والتحليل الوراثي

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

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

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

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

تقييم مساهمات أنواع محددة لتنمية الجمجمة عن طريق الوهم السمان بطة

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