وأيد هذا العمل من قبل المعاهد الوطنية للصحة (R01 - HD044750) ، منحة المهنة من مؤسسة العلوم الوطنية ، وبدعم من جمعية القلب الاميركية (البداية المنح التي تقدم للمساعدة). الكتاب أشكر أعضاء مختبر ديفيدسون : HY كيم ، M. Dassow فون وجيه تشو (للحصول على مساعدة ذوي المسؤوليات مختبر يوميا) ، وتشانغ لين لمساعدتها في مجال البيولوجيا الجزيئية وتوليف مرنا. نحن نعترف بجهود جميع مختبرات تجريبية من الضفدع (وخصوصا راي كيلر ودوغ لDeSimone) الذين ساهموا في جزء من هذا البروتوكول ، من خلال تطوير واختبار طرق مختلفة على مر السنين. نشكر ريتشارد هارلاند والينجفورد جون للهدايا من نوعها ، ذاكرة GFP والبلازميدات moesin - GFP على التوالي.

<ol>
	<li>Ninomiya, H., Winklbauer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+coating+controls+mesenchymal+shape+change+through+tissue-positioning+effects+and+reduction+of+surface-minimizing+tension.">Epithelial coating controls mesenchymal shape change through tissue-positioning effects and reduction of surface-minimizing tension.</a> Nat Cell Biol. 10 (1), 61-61 (2008).</li><li>Kay, B. K., Peng, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Xenopus laevis: Practical Uses in Cell and Molecular Biology. , (1991).</li><li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+eggs+from+Xenopus+laevis+females.">Obtaining eggs from Xenopus laevis females.</a> J Vis Exp. , (2008).</li><li>Cohen, S., Au, S., Pante, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis+oocytes.">Microinjection of Xenopus laevis oocytes.</a> J Vis Exp. , (2009).</li><li>Nieuwkoop, P. D., Faber, 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> Normal Tables of Xenopus laevis (Daudin). , (1967).</li><li>Davidson, L. A., Ezin, A. M., Keller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+wound+healing+by+apical+contraction+and+ingression+in+Xenopus+laevis.">Embryonic wound healing by apical contraction and ingression in Xenopus laevis.</a> Cell Motil Cytoskeleton. 53 (3), 163-163 (2002).</li><li>Joshi, S. D., Dassow, M. v. o. n., Davidson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+control+of+excitable+embryonic+tissues:+three+stimuli+induce+rapid+epithelial+contraction.">Experimental control of excitable embryonic tissues: three stimuli induce rapid epithelial contraction.</a> Exp Cell Res. 316 (1), 103-103 (2010).</li></ol>

الإعلان عن أي تضارب في المصالح.

قدمنا ​​البروتوكولات التجريبية تستخدم بانتظام في المختبر لتغيير صورة الهيكل الخلوي حيوي في القشرة خلية قمية وتتأرجح أغشية الخلايا من الخلايا الظهارية في أجنة القيطم. ينبغي للمرء أن استخدام هذه البروتوكولات كنقطة انطلاق للتصوير يعيش من الأشكال الخلايا الظهارية ؛ الاستفادة المثلى من هذا البروتوكول هو أمر ضروري وبعض الإعدادات سوف تتغير تبعا لنوع النظام المجهر أحد لديه وتهتم نوع من البروتينات واحدة في التصوير . معا ، والخطوات البروتوكول (يزدرع العزلة ، والتصوير والتصور المكونات الخلوية وشبه الخلوية ، ومنهجية القياس الكمي) توفير إجراء دراسة شاملة لالتشكل الظهارة. الجوانب الحاسمة لنتذكر <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> من المهم أن تعد ذات جودة عالية لمرنا الدقيقة الحقن في الأجنة. يتم تخزين أنابيب مرنا على الجليد لتفادي التدهور. اختبار مرة واحدة ، يتم تخزين الأنابيب في -80 درجة مئوية في أصغر aliquots متعددة لتجنب تجميد أذاب دورات. </li><li style=";text-align:right;direction:rtl"> فمن الضروري ضخ مرنا في مواقع متعددة لضمان التوزيع العادل للمرنا أكثر من حقل كبير من الخلايا الحيوانية في الأديم الظاهر الغطاء. </li><li style=";text-align:right;direction:rtl"> إضافة مضاد حيوي / مضاد فطري ضروري للثقافة على المدى الطويل لتثبيط نمو البكتيريا والفطريات. </li><li style=";text-align:right;direction:rtl"> يجب توخي الحذر أثناء الضغط explants تحت شظايا ساترة منذ بأكثر من المطلوب الضغط يمكن سحق explants. </li><li style=";text-align:right;direction:rtl"> بينما التصوير ، وإعدادات الأمثل ضرورية لجمع البيانات القصوى مع نوعية أفضل صورة ممكنة تعكس سلوكيات الخلية المحلية والديناميات البروتين. اختيار الإعدادات المثلى يأتي مع الممارسة والخبرة. </li><li style=";text-align:right;direction:rtl"> لتحليل الصور نوصي بشدة ImageJ. ImageJ هي ممتازة مفتوحة المصدر الأداة التي يمكن تحميلها وتعديلها مع اضافة وحدات الماكرو المخصصة مكتوبة أو تنزيلها والمكونات الإضافية. </li></ol>

live-cell imaging and quantitative analysis of embryonic epithelial cells in xenopus laevis

الخلايا الظهارية الجنينية بمثابة نموذج مثالي لدراسة التشكل حيث متعددة الخلايا الأنسجة تغيرات في الهندسة الخاصة بهم ، مثل التغيرات في المنطقة وارتفاع سطح الخلية الخلية ، وفيها خلايا الخضوع الانقسام والهجرة. وعلاوة على ذلك ، يمكن أيضا تنظيم الخلايا الظهارية الحركات التخلق في الأنسجة المجاورة<sup&gt; 1</sup&gt;. والطريقة التقليدية لدراسة الخلايا والأنسجة الطلائية تنطوي على تثبيت النسيجية والكيميائية وأساليب لتحديد مورفولوجية الخلية أو توطين بروتينات معينة من الفائدة. هذه الأساليب لا تزال مفيدة وتوفر &quot;لقطات&quot; من الأشكال الخلايا والأنسجة العمارة ، ومع ذلك ، لا يزال هناك الكثير يجب أن يفهم حول كيفية الحصول على خلايا محددة الأشكال ، وكيفية نقل أو البروتينات المختلفة لتوطين وظائف محددة ، وما هي الخلايا اتبع المسارات نحو النهائي لها مصير متباينة. ارتفاع القرار يعيش التصوير يكمل الأساليب التقليدية ، ويسمح أيضا مزيد من التحقيق المباشر في العمليات الحيوية الخلوية المشاركة في ، وتشكيل صيانة التشكل ، وصحائف متعددة الخلايا الظهارية. هنا نظهر الطرق التجريبية من العزلة من الأنسجة الحيوانية من قبعة<em&gt; القيطم المورق</em&gt; أجنة لتصوير مبائر من الخلايا الظهارية ونهج القياس البسيطة التي يمكن أن تزيد معا الدراسات الجزيئية والخلوية التشكل الظهارة.

Watch this Scientific Journal Video about Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis at JoVE.com

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

القيطم ظهائر الجنينية هي نموذج مثالي لنظام دراسة سلوكيات الخلية مثل التنمية وتغير شكل القطبية خلال التشكل الظهارة. ويتزايد الأنسجة التقليدية لعينات محددة يجري استكمالها التصوير الخلية الحية مبائر. هنا نظهر طرق لعزل الأنسجة الضفادع وتصور الخلايا الظهارية العيش والهيكل الخلوي وذلك باستخدام الخلايا الحية المجهري متحد البؤر.

تعيش خلية التصوير والتحليل الكمي من الخلايا الظهارية في الجنينية القيطم المورق</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

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Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

14.9K Views.  University of Pittsburgh. القيطم ظهائر الجنينية هي نموذج مثالي لنظام دراسة سلوكيات الخلية مثل التنمية وتغير شكل القطبية خلال التشكل الظهارة. ويتزايد الأنسجة التقليدية لعينات محددة يجري استكمالها التصوير الخلية الحية مبائر. هنا نظهر طرق لعزل الأنسجة الضفادع وتصور الخلايا الظهارية...

Video: Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

البلاستيك وتضمينها Sectioning الجنين المورق القيطم

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

الحصول على البيض من الإناث المورق القيطم

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

إعداد والتقطير من البيض مقتطفات القيطم المورق

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection من البويضات المورق القيطم

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

الكمية خلية حية الإسفار - المجهري تحليل الخميرة الانشطار

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

الاستقراء وتحليل الظهارية الوسيطة الانتقالية ل

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

زرع الوجه في<em&gt; القيطم المورق</em&gt; الأجنة

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

التنميط الفردية الخلايا الجذعية الجنينية البشرية من خلال الكمي RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

القيطم المورق نموذجا لتحديد الترجمة انخفاض قيمة

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...