وقد تم تطوير هذا البروتوكول في إطار منحة يلكوم ترست. نشكر السيدة مورين سينكلير للحفاظ على مخزون الطاير وإعداد الطعام ذبابة على مر السنين. ونود أيضا أن نشكر السيد مايكل Aitchison لمساعدته وتقديم الدعم التقني في البلدان النامية من هذا البروتوكول.

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flies+without+a+spindle+checkpoint.">Flies without a spindle checkpoint.</a> Nat. Cell Biol. 9, 565-572 (2007).</li><li>Tang, Z., Shu, H., Oncel, D., Chen, S., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+Cdc20+by+Bub1+provides+a+catalytic+mechanism+for+APC/C+inhibition+by+the+spindle+checkpoint.">Phosphorylation of Cdc20 by Bub1 provides a catalytic mechanism for APC/C inhibition by the spindle checkpoint.</a> Mol. Cell. 16, 387-397 (2004).</li><li>Huang, J., Raff, J. 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J. 23, 3133-3143 (2004).</li><li>Lorca, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fizzy+is+required+for+activation+of+the+APC/cyclosome+in+Xenopus+egg+extracts.">Fizzy is required for activation of the APC/cyclosome in Xenopus egg extracts.</a> Embo. J. 17, 3565-3575 (1998).</li><li>Howell, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+dynein/dynactin+drives+kinetochore+protein+transport+to+the+spindle+poles+and+has+a+role+in+mitotic+spindle+checkpoint+inactivation.">Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.</a> J. Cell Biol. 155, 1159-1172 (2001).</li><li>Foe, V. E., Alberts, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+nuclear+and+cytoplasmic+behaviour+during+the+five+mitotic+cycles+that+precede+gastrulation+in+Drosophila+embryogenesis.">Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis.</a> Journal of cell science. 61, 31-70 (1983).</li><li>Li, D., Morley, G., Whitaker, M., Huang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+Cdc20+to+the+kinetochore+requires+BubR1+but+not+Mad2+in+Drosophila+melanogaster.">Recruitment of Cdc20 to the kinetochore requires BubR1 but not Mad2 in Drosophila melanogaster.</a> Mol. Cell Biol. 30, 3384-3395 (2010).</li><li>Karess, R. E., Glover, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P+element+mediated+germ+line+transformation+of+Drosophila.">P element mediated germ line transformation of Drosophila.</a> DNA cloning: A practical approach. 2, 121-141 (1985).</li><li>Wakefield, J. G., Huang, J. Y., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosomes+have+a+role+in+regulating+the+destruction+of+cyclin+B+in+early+Drosophila+embryos.">Centrosomes have a role in regulating the destruction of cyclin B in early Drosophila embryos.</a> Current Biology. 10, 1367-1370 (2000).</li><li>Huang, J. Y., Raff, J. 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+dynamic+localisation+of+the+Drosophila+APC/C:+evidence+for+the+existence+of+multiple+complexes+that+perform+distinct+functions+and+are+differentially+localised.">The dynamic localisation of the Drosophila APC/C: evidence for the existence of multiple complexes that perform distinct functions and are differentially localised.</a> Journal of Cell Science. 115, 2847-2856 (2002).</li><li>Raff, J. W., Jeffers, K., Huang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+Fzy/Cdc20+and+Fzr/Cdh1+in+regulating+the+destruction+of+cyclin+B+in+space+and+time.">The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time.</a> Journal of Cell Biology. 157, 1139-1149 (2002).</li><li>Brust-Mascher, I., Scholey, 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=Microinjection+Techniques+for+Studying+Mitosis+in+the+Drosophila+melanogaster+Syncytial+Embryo.">Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo.</a> J. Vis. Exp. (31), e1382 (2009).</li><li>Figard, L., Sokac, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Cell+Shape+Change+in+Living+Drosophila+Embryos.">Imaging Cell Shape Change in Living Drosophila Embryos.</a> J. Vis. Exp. (49), e2503 (2011).</li></ol>

ليس لدينا ما يكشف.

بروتوكول الموصوفة هنا هو طريقة عامة لتصوير الأجنة يطير المخلوي باستخدام TCS لايكا ليزر متحد البؤر SP2 مجهر المسح ويمكن تعديلها لتناسب أنظمة المجهر الأخرى، ويمكن أيضا أن تتكيف مع دراسة وظائف الجينات الأخرى التي تستخدم أجنة ذبابة الفاكهة المخلوي. وقد استخدمنا هذا البروتوكول لدراسة جوانب كثيرة من حاجز المغزل الجمعية، وديناميات البروتين والتحلل البروتيني بروتين باستخدام الذباب المعدلة وراثيا أو الأجسام المضادة بولكلونل لتصور توطين البروتين في عينات حية أو ثابت 4،6،12-14. فمن أبسط للحفاظ على الذباب في قوارير جديدة الغذائية ذبابة عند جمع أعداد صغيرة (~ 10 - 100) من البيض. وهذا يقلل من الازعاج من صنع وإعداد لوحات إضافية أجار عصير التفاح والغرف جمع البيض كما هو موضح في غيرها من المنشورات 15،16. إضافة مربع صغير من زجاج مكسور ساترة في واحدة من نهاية الشريط الغراء على ساترة يجعل من الاسهل بكثيرلفتح إبرة وتحديد حجم حقن عن طريق ضبط إعدادات النظام microinjection بينما الإبرة وتخبط في مجال النفط 10S. درجة جفاف الجنين من المهم لنجاح الحقن في تجنب التسربات والحفاظ على سلامة الأجنة وخاصة لأولئك التجارب التي تحتاج إلى فترة طويلة من الوقت أو عندما رفع transformants بعد الحقن. ومع ذلك، لا توجد قاعدة ذهبية للبالضبط كيف طويل هو مطلوب جفاف. هذا يختلف من تجربة إلى تجربة ويعتمد على مقدار من الحل حقن والرطوبة في بيئة العمل. يمكن التلاعب بها وظائف بروتين معين من الاهتمام من قبل microinjecting مثبطات بروتين معين، أحادية أو بولي نسيلي أجسام مضادة ضد بروتين معين، أو الحمض النووي الريبي رسول الببتيدات fluorescently المسمى تحت نوع البرية أو الخلفيات طفرة جينية. كثير من هذه الخطوط أو متحولة GFP الموسومة خطوط المعدلة وراثيا هي مركبات علناوترد ailable من مراكز الأوراق المالية ذبابة الفاكهة ومعظمها في Flybase ( <a href="http://flybase.org/" target="_blank">http://flybase.org/~~V</a> ) ويمكن البحث على الانترنت.

الأساسية والمعدات والكواشف: مبائر نظام التصوير: نظام التصوير الموصوفة في هذا البروتوكول هو TCS لايكا SP2 ليزر متحد البؤر المسح نظام مجهر مقلوب. الأسلوب هو موضح هو أيضا مناسبة ربما مع بعض التعديلات الطفيفة لأنظمة التصوير الأخرى. تشريح المجهر: نحن نستخدم SZX7 أوليمبوس مع PLAPO 1X DF-4 عدسة. إبرة مجتذب: المشتعلة / براون micropipette مجتذب (سوتر من نموذج، الآلة: P-97). نظام Microinjection: يوصف نظام microinjection إيبندورف لكن يمكن استخدام أي نظام آخر مناسب. يطير موزع الغذاء: Jencons العلمية مضخة تحوي المحدودة. الكواشف: <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> المكونات </td><td> الوزن<html> ق </td><td> موارد </td><td> الكثير رقم </td></tr><tr><td> الذرة وجبة </td><td> 100.0g </td><td> SUMA، المملكة المتحدة </td><td></td></tr><tr><td> السكر البني </td><td> 50.0g </td><td> بيلينغتون، والمملكة المتحدة </td><td></td></tr><tr><td> خميرة جافة </td><td> 25.0g </td><td> DCL خميرة المملكة المتحدة المحدودة. </td><td></td></tr><tr><td> أجار </td><td> 12.5g </td><td> فيشر العلمية </td><td> 106556 </td></tr><tr><td> السوربيك حمض </td><td> 0.4g </td><td> BDH، VWR الدولية المحدودة في المملكة المتحدة </td><td> 8829310 </td></tr><tr><td> بنزويك </td><td> 2.9g </td><td> فيشر العلمية </td><td> 1019599 </td></tr><tr><td> Nipagin (الميثيل-4-هيدرو xybenzoate) </td><td> 0.9g </td><td> BDH، VWR الدولية المحدودة في المملكة المتحدة </td><td> K35969015 </td></tr><tr><td colspan="3"> H 2 O يصل الى 1L </td><td></td></tr></tbody></table>الطبقة = &quot;jove_content&quot;&gt; الجدول رقم 1. تطير المكونات الغذائية. Holocarbon 700 و 27 الزيوت التي تم شراؤها من شركة سيجما. خميرة جافة: توماس أليسون، المملكة المتحدة. إعداد الغراء هيبتان: خذ حوالي 1.5 متر من الشريط سكوتش على الوجهين، ووضعها في أنبوب الصقر 15ml مع 5ml من هيبتان وتدويره لمدة 3-5 ساعات أو بين عشية وضحاها. بعد ذلك الوقت انقسم الى 4 aliquots على قدم المساواة في 1.5 مل من أنابيب الطرد المركزي إيبندورف وتدور لمدة 5 دقائق في وقت سريع في جهاز للطرد المركزي مقاعد البدلاء أعلى لإزالة أي حطام. تخزين حل الغراء هيبتان في القارورة الحجمية 10ML والحفاظ على لاستخدامها. فإن قوة الغراء تختلف وفقا لتركيز وكميات من الصمغ المستخدمة. عادة، وأرق الغراء تنتج أقل خلفية صاخبة عندما تم فحصها بواسطة المجهر متحد البؤر. <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ساترة: BDH، VWR الدولية المحدودة في المملكة المتحدة </li><li style=";text-align:right;direction:rtl"> شريحة المجهر: BDH، VWR الدولية المحدودة في المملكة المتحدة &lt;/ لى&gt; <li style=";text-align:right;direction:rtl"> Siliconised زجاج الشعيرات الدموية: GC-100-10، في جامعة هارفارد </li><li style=";text-align:right;direction:rtl"> غرامة غيض التحميل: 20μl إيبندورف الكثير: W215818J </li><li style=";text-align:right;direction:rtl"> ملاقيط </li><li style=";text-align:right;direction:rtl"> غرامة القلم الفرشاة </li></ul>

studying mitotic checkpoint by illustrating dynamic kinetochore protein behavior and chromosome motion in living drosophila syncytial embryos

حاجز الجمعية المغزل (SAC) آلية هي إشارة نشط، والذي يراقب التفاعل بين kinetochores كروموسوم وميكروتثبول المغزل للحيلولة دون ظهور طور الصعود حتى ترتبط بشكل صحيح الكروموسومات. الخلايا استخدام هذه الآلية لمنع عدم الاستقرار أو عدم توازن الصبغيات الجينية، وبالتالي السرطان والأمراض البشرية الأخرى مثل العيوب الخلقية ومرض الزهايمر 1. وهناك عدد من المكونات مثل SAC Mad1، Mad2، Bub1، BubR1، Bub3، Mps1، Zw10، وقد تم تحديد ورود أورورا كيناز باء، وأنهم جميعا البروتينات kinetochore الدينامية (2). تشير الدلائل إلى أن kinetochore هو المكان الذي بدأ إشارة ساك. الهدف الرئيسي ساك التنظيمية Cdc20. Cdc20 هي واحدة من وسائل تفعيل APC / C (أ ف naphase romoting C omplex أو yclosome C) (3) وأساسي هو أيضا بروتين kinetochore دينامية 4-6. عند تفعيلها، وSAC يثبط من نشاط وPC / C لمنع تدمير اثنين من ركائز رئيسية، cyclin باء وsecurin، مما يحول دون انتقال الطورية إلى طور الصعود 7،8. بالضبط كيف يتم بدء إشارة SAC وتجميعها على kinetochores ونقلت على لآسيا والمحيط الهادئ / C لمنع وظيفتها لا تزال بعيدة المنال. ذبابة الفاكهة هو نظام لين العريكة جدا التجريبي؛ كائن أبسط من ذلك بكثير وأفضل مفهومة مقارنة مع واحد ولكن الإنسان الذي أسهم في العمليات الأساسية المشتركة. هو، ربما، واحدة من أفضل الكائنات الحية لاستخدامها في التصوير دراسات البيولوجية في الخلايا الحية، وخاصة بالنسبة للالتصور للأحداث الإنقسامية في المكان والزمان، كما أن الجنين في وقت مبكر يذهب من خلال 13 دورات السريع تقسيم النووية بشكل متزامن (8-10 دقائق لكل دورة على درجة مئوية 25)، وتنظم تدريجيا نوى في أحادي الطبقة واحدة فقط تحت القشرة 9. هنا، أقدم طريقة الحيوي التصوير باستخدام المعدلة وراثيا ذبابة الفاكهة EXPRESالغناء GFP (الأخضر بروتين فلوري) أو البديل لها استهداف البروتينات من الفائدة وTCS لايكا SP2 نظام ليزر متحد البؤر مجهر المسح لدراسة وظيفة SAC في الذباب، من جراء عرض صور من البروتينات الانصهار GFP من بعض المكونات SAC، Cdc20 وMad2 ، كما في المثال.

Watch this Scientific Journal Video about Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos at JoVE.com

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

وkinetochore هو المكان الذي يبدأ SAC الاشارة رصد الفصل الإنقسامية من chromatids شقيقة. تم وصف طريقة لتصور التوظيف ودوران واحد من البروتينات kinetochore والتنسيق مع الحركة في كروموسوم<em&gt; ذبابة الفاكهة</em&gt; أجنة باستخدام نظام المسح الضوئي ليزر متحد البؤر لايكا.

دراسة حاجز الميتوزى بواسطة يوضح الحيوي Kinetochore السلوك البروتين والحركة كروموسوم في المعيشة<em&gt; ذبابة الفاكهة</em&gt; الجنين المخلوي

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

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Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

11.3K Views.  University of Newcastle, United Kingdom. وkinetochore هو المكان الذي يبدأ SAC الاشارة رصد الفصل الإنقسامية من chromatids شقيقة. تم وصف طريقة لتصور التوظيف ودوران واحد من البروتينات kinetochore والتنسيق مع الحركة في كروموسوم ذبابة الفاكهة أجنة باستخدام نظام المسح الضوئي ليزر متحد البؤر لايكا.

Video: Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

تطبيق فاتح الشفاء الراتنج لاصق الأسنان أو تركيب لأقطاب مجسات Microdialysis في التجارب المزمنة

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

عالية الدقة قياس سلوك رائحة يحركها يرقات ذبابة الفاكهة في

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

تقنيات Microinjection لدراسة الانقسام الميتوزي في ذبابة الفاكهة السوداء البطن الأجنة المخلوي

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

داخل الجسم الحي الطرد المركزي الجنين ذبابة الفاكهة

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

رني التدخل عن طريق الحقن في الرنا المزدوج الجديلة ذبابة الفاكهة الأجنة

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

التصوير تغيير الشكل في الخلية الحية ذبابة الفاكهة الأجنة

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

تحديد البروتين البروتين التفاعل في<em&gt; ذبابة الفاكهة</em&gt; رؤساء الكبار حسب جنبا الى جنب الانجذاب تنقية (وات)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

و في الجسم الحي يشابك النهج لعزل البروتين المجمعات من ذبابة الفاكهة الأجنة

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

الجينوم على نطاق فحص التفاعلات بين البروتينات التي كتبها البروتين جزء التكملة الفحص (PCA) في الخلايا الحية

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

دراسة الميتوكوندريا هيكل وظيفة في<em&gt; ذبابة الفاكهة</em&gt; المبايض

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...