ويرعى الأبحاث في المختبر من قبل المركز الإنترنت سوكول الوطنية للصحة.

1. الإخصاب في المختبر من البيض القيطم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الحصول على البيض من الضفادع الإناث التي حقنت مع موجهة الغدد التناسلية المشيمية البشرية (400 وحدة / الضفدع) 12 ساعة قبل التجربة. </li><li style=";text-align:right;direction:rtl"> البيض في المكان على كمية صغيرة (0.5-1 مل) من 1 × مارك محلول رينغر تعديل (MMR) 10 إلى البيض وتخصيبها في المختبر منهم بقطعة صغيرة من الخصية تشريح. بعد 2-3 دقيقة ، إضافة 0.1 X MMR لتغطية كامل سطح البيض. في 20 دقيقة ، يتم إزالة البيض هلام معطف بواسطة السيستين 3 ٪ -- HCL (معدلة لدرجة الحموضة (8) مع هيدروكسيد الصوديوم). يتم غسل البيض بنسبة 0.1 X ثلاث مرات MMR وغادرت في حاضنة الباردة (13 درجة مئوية) للحقن. </li><li style=";text-align:right;direction:rtl"> ويسمح لوضع البويضات المخصبة إلى مرحلة الخلية 2-4. عن الحقن ، ويتم نقل الأجنة في محلول يحتوي على 3 ٪ Ficoll ، 0.5 X MMR. </li></ol> 2. RNA Microinjection <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تم تجميعها من الرناوات قوالب باستخدام الحمض النووي خطي mMessage mMachine مجموعة (Ambion) والمخفض مع ريبونوكلياز خالية من المياه على تركيز المخزون من 0،1-1 ميكروغرام / ميكرولتر. ويتم تحديد الجرعات المثلى من RNAs للحقن في التجارب الرائدة. وتستخدم لالرناوات Diversin - RFP ، علامة غشاء GFP - CAAX وFrizzled 8 في 0،1-1 نانوغرام في الحقن. </li><li style=";text-align:right;direction:rtl"> إبر الحقن مستعدون مع مجتذب إبرة من شعري ثم مع طاحونة الإبرة. قبل الحقن ، ويتم معايرة كل إبرة مع الماء لطرد 10 لحقن السائل NL الواحد. </li><li style=";text-align:right;direction:rtl"> عن الحقن ، وتوضع على طبق الأجنة البلاستيكية إلى قطرات كبيرة من Ficoll 3 ٪ ، 0.5 X MMR. يرشح احد ميكروليتر من محلول الحمض النووي الريبي في إبرة الحقن مع microinjector Narishige. يتم حقن 10 NL من الحمض النووي الريبي في الأجنة الحيوانية blastomeres الخلية أغسطس 2-3 مرات. يتم نقل الأجنة في بئر حقن من لوحة ال 12 أيضا. </li></ol> 3. إعداد Explants الأدمة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عندما تصل إلى حقن الأجنة المبكرة مرحلة المعيدة ، يتم نقلهم الى حل MMR 0.6 x في طبق من البلاستيك 3 سم مغلفة agarose 1 ٪. تتم إزالة الغشاء المحي يدويا مع زوج من الملقط. يتم رفعه explants الأدمة من أجنة باستخدام إبرة التنغستن وhairloop ملف. </li><li style=";text-align:right;direction:rtl"> يتم نقل explants الأدمة في قارورة زجاجية وثابتة مع الفورمالدهايد 3.7 ٪ في الفوسفات مخزنة المالحة (PBS) لمدة 30 دقيقة. تغسل explants ثابت مع ثلاث مرات في برنامج تلفزيوني (10 دقيقة لكل منهما). يتم تضمين دابي في ثالث لغسل وصمة عار النوى. </li><li style=";text-align:right;direction:rtl"> هي التي شنت على الزجاج Explants الشريحة. وترد قطعتين من الشريط سكوتش إلى الشريحة ويتم وضع شرائط explants بين البلدين. منذ السطح الخارجي للexplants غير مصطبغة ، يتعين على الجانب الداخلي من explants مواجهة الهدف المجهر. إضافة 2-3 قطرات 20 ميكرولتر من الحل تركيب (70 ٪ الجلسرين في برنامج تلفزيوني من بينهم 25 ملغ / مل DABCO ومعاداة يتلاشى الكاشف) 11. وضع ساترة على القمة. </li></ol> 4. تصوير Explants تحت المجهر نيون <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وينظر العينات تحت المجهر Axioplan زايس الفلورسنت مع الفلاتر المناسبة. </li><li style=";text-align:right;direction:rtl"> يتم التقاط صور مع المرفق Apotome لتصور محدد من طائرة explants مستقلة عديدة. </li></ol> 5. Cryosectioning Cryosectioning هو وسيلة بديلة لتصور توزيع البروتينات الفلورية في الخلية وأكثر قابلية للتطبيق لimmunostaining. في المرحلة 10 ، والأجنة ثابتة لمدة 1-2 ساعات مع لتثبيتي دنت (20 ٪ DMSO ، و 80 ٪ الميثانول) ، وغسلها مع برنامج تلفزيوني ، وجزءا لا يتجزأ من حل 15 ٪ سكروز gelatin/15 الأسماك 11 ٪. يتم تجميد الأجنة بسرعة مضمن على الثلج الجاف ويتم إنشاؤها على cryosections ناظم البرد لايكا. والمقاطع العرضية وتشمل خلايا الأدمة التي ترث الرناوات حقن البروتين ومنتجاتها ترجمتها. المقاطع الاحتفاظ مضان ويمكن immunostained مع الاجسام المضادة المحددة ، ثم المسمى مع الأجسام المضادة الثانوية مترافق مع مضان. هي ملطخة النوى مع دابي. وسائل الإعلام المتصاعدة هي نفسها كما هو موضح أعلاه. لا يمكن أن يؤديها التصوير على النحو المبين أعلاه. 6. ممثل النتائج : <img src="/files/ftp_upload/2700/2700fig1.jpg" alt="الشكل 1" /> الشكل 1. مستقبلات Frizzled المجندين Diversin إلى غشاء الخلية. خلايا الأدمة معربا عن 8 Frizzled (Xfz8) والرناوات الدرجة الدرجة تكشف - RFP - RFP في غشاء الخلية ، وبدلا من الجسيم المركزي (كما يتضح من ز تويولين تلطيخ المشترك). مخطط التجربة في أعلى ؛ يتم عرض نموذجي عبر الباب.

<ol>
	<li>Gurdon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+induction+---+molecular+aspects.">Embryonic induction --- molecular aspects.</a> Development. 99, 285-306 (1987).</li><li>Moody, 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> Principles of Developmental Genetics. , (2007).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+in+development+and+disease.">Wnt/beta-catenin signaling in development and disease.</a> Cell. 127, 469-480 (2006).</li><li>Klaus, A., Birchmeier, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signalling+and+its+impact+on+development+and+cancer.">Wnt signalling and its impact on development and cancer.</a> Nat Rev Cancer. 8, 387-398 (2008).</li><li>Gordon, M. D., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling:+multiple+pathways,+multiple+receptors,+and+multiple+transcription+factors.">Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors.</a> J Biol Chem. 281, 22429-22433 (2006).</li><li>Schwarz-Romond, T., Asbrand, C., Bakkers, J., Kuhl, M., Schaeffer, H. J., Huelsken, J., Behrens, J., Hammerschmidt, M., Birchmeier, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ankyrin+repeat+protein+Diversin+recruits+Casein+kinase+Iepsilon+to+the+beta-catenin+degradation+complex+and+acts+in+both+canonical+Wnt+and+Wnt/JNK+signaling.">The ankyrin repeat protein Diversin recruits Casein kinase Iepsilon to the beta-catenin degradation complex and acts in both canonical Wnt and Wnt/JNK signaling.</a> Genes Dev. 16, 2073-2084 (2002).</li><li>Moeller, H., Jenny, A., Schaeffer, H. J., Schwarz-Romond, T., Mlodzik, M., Hammerschmidt, M., Birchmeier, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversin+regulates+heart+formation+and+gastrulation+movements+in+development.">Diversin regulates heart formation and gastrulation movements in development.</a> Proc Natl Acad Sci U S A. 103, 15900-15905 (2006).</li><li>Itoh, K., Jenny, A., Mlodzik, M., Sokol, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosomal+localization+of+Diversin+and+its+relevance+to+Wnt+signaling.">Centrosomal localization of Diversin and its relevance to Wnt signaling.</a> J. Cell Sci. 122, 3791-3798 (2009).</li><li>Itoh, K., Jacob, J., Sokol, Y. S. <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+Xenopus+Frizzled+8+in+dorsal+development.">A role for Xenopus Frizzled 8 in dorsal development.</a> Mech Dev. 74, 145-157 (1998).</li><li>Newport, J., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+developmental+transition+in+early+Xenopus+embryos:+I.+characterization+and+timing+of+cellular+changes+at+the+midblastula+stage.">A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage.</a> Cell. 30, 675-686 (1982).</li><li>Itoh, K., Brott, B. K., Bae, G. U., Ratcliffe, M. J., Sokol, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+localization+is+required+for+Dishevelled+function+in+Wnt/beta-catenin+signaling.">Nuclear localization is required for Dishevelled function in Wnt/beta-catenin signaling.</a> J Biol. 4, 3-3 (2005).</li></ol>

الإعلان عن أي تضارب في المصالح.

لقد استخدمنا بروتوكول أعلاه لتميز الترجمة في Diversin التحت خلوية. explants في القطب الحيواني ، تم الكشف عن Diversin - RFP بجوار النواة وcolocalized تويولين مع الجميع ، علامة centrosomal ، cryosections في (الشكل 1). حالما يتم التعرف على توطين التحت خلوية من البروتين ، يمكن إنشاء بنيات الحذف لإنشاء المجالات التي البروتين ضرورية وكافية لتوطين التحت خلوية. باستخدام هذا النهج ، فقد تم تعيين مجالات الترجمة من centrosomal Diversin إلى وسط وفي المجالات كربوكسي محطة من البروتين ، وكلاهما يحتوي على لفائف ملفوف عزر 8. ويمكن استخدام البروتوكول نفسه في الدراسات ، التي غيرت التعريب البروتين ردا على الإشارة. وجدنا أن تفرز بروتينات Wnt العمل على نقل فرقة المشاة - RFP للهياكل المنقط المتاخمة لغشاء الخلية ، في حين Fz8 المجندين الدرجة - RFP إلى بقع غشاء الخلية (الشكل 1). اكتشفنا كذلك أن المجال كربوكسي المحطة ليست عنصرا أساسيا للتوظيف الغشاء في حد ذاتها ، ولكن المطلوب للتجنيد الغشاء المستقطب. باختصار ، فإن بروتوكول أعلاه تجريبية مساعدة في دراسات متنوعة من البروتين البروتين التفاعلات وتوطين البروتين لمقصورات الخلوية المختلفة بعد تنشيط الخلايا مع عوامل نمو معينة أو بروتينات الإشارة.

polarized translocation of fluorescent proteins in xenopus ectoderm in response to wnt signaling

قطبية الخلية هي الخاصية الأساسية للخلايا حقيقية النواة التي يتم تنظيمها بشكل حيوي على حد سواء بفعل عوامل ذاتية وخارجي أثناء التطور الجنيني 1 و 2. واحد من مسارات إشارات تشارك في هذا النظام هو الطريق Wnt ، والذي يستخدم عدة مرات خلال مرحلة التطور الجنيني والحرجة لأمراض الإنسان 3 ، 4 ، 5. المكونات الجزيئية متعددة من هذا الممر تنظيم coordinately يشير بطريقة مكانيا المقيدة ، ولكن لا يفهم الآليات الكامنة بشكل كامل. القيطم طلائي الخلايا الجنينية هو نظام ممتاز لدراسة توطين التحت خلوية من مختلف البروتينات الإشارة. يتم التعبير عن البروتينات الفلورية الانصهار في الأجنة القيطم microinjection بواسطة الحمض النووي الريبي ، مستعدون explants الأدمة ويتم تقييم الترجمة البروتين epifluorescence. في هذا البروتوكول التجريبي نحن تصف كيفية الترجمة من Diversin التحت خلوية ، وهو بروتين حشوية التي تورطت في الإشارات وتحديد الخلية القطبية 6 ، 7 هو تصور في خلايا الأدمة القيطم لدراسة Wnt نقل الإشارة 8. Coexpression ليجند Wnt أو مستقبلات Frizzled يغير توزيع Diversin تنصهر مع بروتين فلوري الأحمر ، RFP ، والمجندين لغشاء الخلية بطريقة الاستقطاب 8 و 9. ينبغي لهذا البروتوكول فيفو السابقين تكون إضافة مفيدة للدراسات في المختبر من خلايا الثدييات مثقف ، والتي تتحكم المكاني للإشارة يختلف من الأنسجة سليمة وأكثر صعوبة في تحليلها.

Watch this Scientific Journal Video about Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling at JoVE.com

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

أصبح القيطم الجنينية الأديم الظاهر نموذجا جذابا للدراسات القطبية الخلية. وصفت والفحص ، التي يتم فيها توزيع التحت خلوية المقررة من البروتينات الفلورية في خلايا الأديم الظاهر. وسوف يساعد هذا البروتوكول تعالج المسائل المتعلقة بمكافحة المكاني للإشارات.

الاستقطاب إزفاء من البروتينات في نيون القيطم الأدمة في الاستجابة لاشارة Wnt

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

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Nanyang Technological University

Research

JoVE Journal

Biology

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

10.7K Views.  Mount Sinai School of Medicine. أصبح القيطم الجنينية الأديم الظاهر نموذجا جذابا للدراسات القطبية الخلية. وصفت والفحص ، التي يتم فيها توزيع التحت خلوية المقررة من البروتينات الفلورية في خلايا الأديم الظاهر. وسوف يساعد هذا البروتوكول تعالج المسائل المتعلقة بمكافحة المكاني للإشارات.

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

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

تصور التعريب في الحمض النووي الريبي القيطم البويضات

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

الكمي بؤر γH2AX في الاستجابة للإشعاع المؤين

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

أعربت المشبك التصحيح وتقنيات الإرواء لدراسة في قنوات ايون القيطم البويضات

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

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 ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

حيوي داخلي المجهري للهياكل التصوير التحت خلوية في الفئران لايف تعرب عن البروتينات الفلورية

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

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. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

ال<em&gt; القيطم</em&gt; سيت قص مفتوحة الفازلين جاب الجهد المشبك تقنية مع التألق

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

إعادة تشكيل β-catenin تدهور في<em&gt; القيطم</em&gt; مستخلصات البيض

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

Quantification of Orofacial Phenotypes in Xenopus

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Quantification of Orofacial Phenotypes in Xenopus

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

الكمي من الظواهر في الوجهية<em&gt; القيطم</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

قياس الاستجابة التغذية الناجم عن الجلوتاثيون في حيدرة

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...