يشكر المؤلفون أعضاء مختبر فيراكسا على تعليقات مفيدة حول المخطوطة واقتراحات لتحسين البروتوكول. تم دعم أف من قبل المعاهد الوطنية للصحة منح GM105813 و NS096402. وقد دعم لي من جامعة ماساتشوستس بوسطن سانوفي جنزيمي الدكتوراه زمالة.

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stringent+analysis+of+gene+function+and+protein-protein+interactions+using+fluorescently+tagged+genes.">Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes.</a> Genetics. 190 (3), 931-940 (2012).</li><li>Veraksa, A., Bauer, A., Artavanis-Tsakonas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein+complexes+in+Drosophila+with+tandem+affinity+purification-mass+spectrometry.">Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry.</a> Dev Dyn. 232 (3), 827-834 (2005).</li><li>Mukherjee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Notch+signalling+by+non-visual+beta-arrestin.">Regulation of Notch signalling by non-visual beta-arrestin.</a> Nat Cell Biol. 7 (12), 1191-1201 (2005).</li><li>Gilbert, M. M., Tipping, M., Veraksa, A., Moberg, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Screen+for+Conditional+Growth+Suppressor+Genes+Identifies+the+Drosophila+Homolog+of+HD-PTP+as+a+Regulator+of+the+Oncoprotein+Yorkie.">A Screen for Conditional Growth Suppressor Genes Identifies the Drosophila Homolog of HD-PTP as a Regulator of the Oncoprotein Yorkie.</a> Dev Cell. 20 (5), 700-712 (2011).</li><li>Anjum, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Toll+Signaling+and+Inflammation+by+beta-Arrestin+and+the+SUMO+Protease+Ulp1.">Regulation of Toll Signaling and Inflammation by beta-Arrestin and the SUMO Protease Ulp1.</a> Genetics. 195 (4), 1307-1317 (2013).</li><li>Degoutin, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riquiqui+and+minibrain+are+regulators+of+the+hippo+pathway+downstream+of+Dachsous.">Riquiqui and minibrain are regulators of the hippo pathway downstream of Dachsous.</a> Nat Cell Biol. 15 (10), 1176-1185 (2013).</li><li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecdysone+receptor+coactivator+Taiman+links+Yorkie+to+transcriptional+control+of+germline+stem+cell+factors+in+somatic+tissue.">The ecdysone receptor coactivator Taiman links Yorkie to transcriptional control of germline stem cell factors in somatic tissue.</a> Dev Cell. 34 (2), 168-180 (2015).</li><li>Lemmon, M. A., Schlessinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+signaling+by+receptor+tyrosine+kinases.">Cell signaling by receptor tyrosine kinases.</a> Cell. 141 (7), 1117-1134 (2010).</li><li>Vincent, J. P., Girdham, C. H., O'Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-autonomous,+ubiquitous+marker+for+the+analysis+of+Drosophila+genetic+mosaics.">A cell-autonomous, ubiquitous marker for the analysis of Drosophila genetic mosaics.</a> Dev Biol. 164 (1), 328-331 (1994).</li><li>Zhai, B., Villen, J., Beausoleil, S. A., Mintseris, J., Gygi, S. 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H., Peng, J., Mayor, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+strategy+to+isolate+ubiquitin+conjugates+reveals+wide+role+for+ubiquitination+during+neural+development.">A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development.</a> Mol Cell Proteomics. 10 (5), (2011).</li><li>Ramirez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Analysis+of+the+Ubiquitin+Landscape+in+the+Drosophila+Embryonic+Nervous+System+and+the+Adult+Photoreceptor+Cells.">Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells.</a> PLoS One. 10 (10), 0139083 (2015).</li></ol>

الكتاب ليس لديهم ما يكشف.

بروتوكول المقدمة هنا هو إجراء بسيط وعمومي لإعداد أقفاص السكان ذبابة الفاكهة على نطاق متوسط، وجعل مقتطفات البروتين خلية كاملة من الأجنة. ويمكن استخدام مقتطفات الناتجة في مجموعة متنوعة من التطبيقات المصب، مثل تنقية المجمعات البروتين على راتنجات تقارب. فمن الأهم...

الشاشات الجينية، ومؤخرا، النهج الجينومية ثورة في دراسة الوظائف البيولوجية. ومع ذلك، يتم ترميز المعلومات الخلوية الهامة في البروتينات ومجموعتها من الشركاء المتفاعلين. في حين أن شاشات المعدل الجيني التقليدية يمكن تحديد مكونات مسار الحد من معدل واستعادة التفاعلات غير المباشرة، وقوة النهج البروتين يكمن في قدرتها على تحديد شبكات التفاعل الفوري الكامل للبروتينات ذات الاهتمام. وبالتالي، فإن بروتيوميكس طريقة متعامدة قيمة لدراسة النظم البيولوجية، ويكمل علم الجينوم، ترانسكريبتوميكس، والشاشات الوراثية التقليدية. وقد أثبتت تقارب مطياف الكتلة تنقية (أب-مس) أن يكون نهجا قويا لدراسة التفاعلات البروتين البروتين (بي) في بيئتهم الأصلية في الخلايا والأنسجة 1 ، 2 . وتسمح هذه الطريقة بتحديد التفاعلات المباشرة أو غير المباشرة في تطوير معينمراحل أو سياقات الأنسجة، وقد استخدمت بنجاح لتحديد متعددة مؤشرات الأداء القياسية الرواية في مجموعة متنوعة من المسارات التنموية (استعرض في المرجع 1 ). على الرغم من النجاح الذي لا جدال فيه لدراسات مؤشر أسعار المنتجين، معظمها قد نفذت في الخلايا المستزرعة، حيث تم الإفراط في التعبير عن البروتينات &quot;الطعم&quot; من الفائدة. هناك نوعان من القضايا مع دراسة مؤشر أسعار المنتجين في زراعة الخلايا: أولا، قد لا يوفر خط خلية محددة مجموعة كاملة من التفاعلات بسبب عدم التعبير عن بعض البروتينات. ثانيا، أوفيركسريسيون عالية عادة ما تستخدم في مثل هذه التحليلات قد تؤدي إلى القطع الأثرية مثل بروتين ميسفولدينغ أو تحديد تفاعلات إيجابية كاذبة. كل من هذه القيود يمكن التغلب عليها من خلال تحليل مؤشر أسعار المنتجين في الجسم الحي . وهناك خطوة محدودة في مثل هذه التجارب هو توافر المواد انطلاق لتنقية المجمعات البروتين. وقد استخدمت منذ فترة طويلة ذبابة الفاكهة ميلانوغاستر كنموذج لتحليل وظيفيوقد ثبت مؤخرا أنه نظام ممتاز لدراسة مؤشرات أسعار المنتجين في الجسم الحي . يمثل تجنين ذبابة الفاكهة نوع الأنسجة جذابة بشكل خاص لدراسة مؤشر أسعار المنتجين، لأن الأجنة يمكن جمعها بسهولة بكميات كبيرة، وأيضا لأن معظم الجينات (&gt; 88٪) يتم التعبير عن مسار التطور الجنيني، وبالتالي توفير غنية في بيئة الجسم الحي للكشف عن ذات الصلة مؤشر أسعار المنتجين 3 . تقليديا، والدراسات البيوكيميائية في الذباب تستخدم جدا على نطاق واسع مجموعات الجنين (100-150 ز)، مثل تلك اللازمة لتنقية ليسيتس النسخي وظيفية 4 ، 5 . الدراسات السابقة أب- مس في ذبابة الفاكهة تحتاج أيضا كميات كبيرة من الأجنة (5-10 ز)، لأنها اعتمدت على نهج تنقية من خطوتين مثل جنبا إلى جنب تنقية تقارب (تاب)، مع فقدان المرتبطة من المواد في كل خطوة 6 . كبير أمونتيسي من المواد البدء في إنشاء مجموعات الجنين في أقفاص سكانية كبيرة، والتي يمكن أن تكون على حد سواء باهظة الثمن (عند شراؤها تجاريا) وتستغرق وقتا طويلا للحفاظ على ونظيفة 7 ، 8 ، 9 . التطورات الأخيرة في تطوير نهج تنقية تقارب خطوة واحدة، فضلا عن حساسية متزايدة من مطياف الكتلة، وخفضت الكمية اللازمة من المواد بدءا من قبل حجم من الحجم. باستخدام علامات مثل الببتيد ستريبتافيدين ملزم (سبب) أو بروتين الفلورسنت الأخضر (غفب) والبدء من أقل من 1 غرام من الأجنة، فمن الممكن لعزل كميات من البروتين الطعم والتفاعل المكونات التي ستكون كافية لتحديد من قبل مطياف الكتلة 10 ، 11 . الهدف من البروتوكول المعروضة هنا هو مساعدة الباحثين أوفيركولي حاجز ينظر إلى التحليل البيوكيميائية من مؤشر أسعار المنتجين في الجسم الحي . تحقيقا لهذه الغاية، ونحن نقدم إجراء بسيط وغير مكلفة لجمع الأجنة ذبابة الفاكهة على نطاق متوسط ​​(0.5-1 ز)، تليها خطوة واحدة إعداد مقتطفات البروتين كامل الخلية التي هي مناسبة لتحليل لاحق من قبل أب-مس أو نهج أخرى . يعتمد أسلوبنا على استخدام 1-L أو 5-L أقفاص السكان حسب الطلب التي يمكن أن تنتج بسهولة من قبل أي مختبر. وعلاوة على ذلك، تم التحقق من صحة ظروف الاستخراج المعروضة هنا في العديد من الدراسات، سواء في الخلايا المستزرعة وفي الجسم الحي 10 ، 12 ، 13 ، 14 ، 15 ، 16 ، 17 .

<table border="0" cellpadding="0" cellspacing="0" width="1384">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Leaktite 5-Qt. Natural Multi Mix Container (Pack of 3)</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:119px;">209330</td>
			<td style="width:644px;">For making 5-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Leaktite 5-Qt. Natural Multi Mix Lid (Pack of 3)</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:119px;">209320</td>
			<td style="width:644px;">Diameter 8.37 in. Lids for 5-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fisherbrand Petri dishes with clear lid, 100 x 15 mm</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875712</td>
			<td style="width:644px;">Material: Polystyrene. To be used with 1-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fisherbrand Petri dishes with clear lid, 150 x 15 mm</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875714</td>
			<td style="width:644px;">Material: Polystyrene. To be used with 5-L cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fisherbrand Petri dishes with clear lid, 60 x 15 mm</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875713A</td>
			<td style="width:644px;">Material: Polystyrene. Used during embryo dechorionation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Sefar NITEX nylon mesh</td>
			<td style="width:183px;">Sefar</td>
			<td style="width:119px;">03-180/44</td>
			<td style="width:644px;">180 micron, 44% open area, used for fly cages.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Milwaukee 3 in. Hole Dozer Hole Saw with Arbor</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:119px;">49-56-9670</td>
			<td style="width:644px;">3 inch cutting tool for making 1-L cages.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">Nalgene Wide-Mouth Straight-Sided PMP Jars with White Polypropylene Screw Closure</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">11-823-33</td>
			<td style="width:644px;">For making 1-L cages. Thermo Scientific cat # 21171000. Alternative 1-L containers can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Red Star Active Dry Yeast, 2 pound pouch</td>
			<td style="width:183px;">Red Star&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:644px;">can be purchased from Amazon.com or other suppliers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Frozen 100% Apple Juice Concentrate&#160;</td>
			<td style="width:183px;"></td>
			<td style="width:119px;"></td>
			<td style="width:644px;">can be purchased from a grocery store. Has to say &#34;100% apple juice&#34; on the can.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Methyl 4-hydroxybenzoate</td>
			<td style="width:183px;">Acros Organics</td>
			<td style="width:119px;">AC126965000</td>
			<td style="width:644px;">also known as methylparaben or Tegosept. Used as a preservative in AJ plates.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">IGEPAL CA-630 for molecular biology, 100 ml</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:119px;">I8896</td>
			<td style="width:644px;">used for preparing lysis buffer.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:439px;">Nalgene Rapid-Flow Sterile Disposable Filter Units with CN Membrane&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">09-740-2A</td>
			<td style="width:644px;">0.2 &#956;m pore size. Used for filtering 5x lysis buffer. Thermo Scientific cat # 1260020.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">CO-RO ROCHE cOmplete Protease Inhibitor Cocktail</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:119px;">11697498001</td>
			<td style="width:644px;">Vial of 20 tablets. Used for protease inhibition in lysis buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Corning SFCA syringe filters&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">09-754-21</td>
			<td style="width:644px;">SFCA membrane, diameter 26 mm, pore size 0.45 &#956;m. Used for filtering final extract samples.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">BD&#160;Luer-Lok Disposable Syringes without Needles</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">14-823-2A</td>
			<td style="width:644px;">for filtering final extract samples. BD cat # 309604.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:439px;">Bleach</td>
			<td style="width:183px;">Clorox</td>
			<td style="width:119px;"></td>
			<td style="width:644px;">used for embryo dechorionation at 50% (vol/vol) in water, can be purchased at any supermarket. It is important to use the Clorox brand, as other brands may result in incomplete dechorionation or may be toxic for embryos.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Corning Costar Netwell Plates</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-213</td>
			<td style="width:644px;">mesh containers for embryo collections. 74 &#956;m mesh size, 6-well.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Wheaton Dounce Tissue Grinders, capacity 15 ml</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">06-435B</td>
			<td style="width:644px;">homogenizer for embryos, with loose and tight pestles. Wheaton cat # 357544.</td>
		</tr>
	</tbody>
</table>

medium-scale preparation of drosophila embryo extracts for proteomic experiments

أصبح تحليل التفاعلات البروتينية البروتين نهجا لا غنى عنه لدراسة العمليات والآليات البيولوجية، مثل إشارات الخلية، وتطوير الكائن الحي، والمرض. وغالبا ما يكون من المرغوب فيه للحصول على معلومات مؤشر أسعار المنتجين باستخدام في الجسم الحي المواد، للحصول على وجهة نظر الأكثر طبيعية وغير منحازة من شبكات التفاعل. ذبابة الفاكهة ذبابة الفاكهة ميلانوغاستر هو منصة ممتازة لدراسة مؤشر أسعار المنتجين في الجسم الحي ، ويتيح نفسه لنهج واضحة لعزل المواد للتجارب البيوكيميائية. على وجه الخصوص، تمثل الأجنة ذبابة الفاكهة نوع مناسب من الأنسجة لدراسة مؤشر أسعار المنتجين، ويرجع ذلك إلى سهولة جمع الحيوانات في هذه المرحلة التنموية وحقيقة أن غالبية البروتينات وأعرب في التطور الجنيني، وبالتالي توفير بيئة ذات صلة للكشف عن معظم مؤشر أسعار المنتجين. هنا نقدم بروتوكول لجمع الأجنة ذبابة الفاكهة على نطاق متوسط ​​(0.5-1 ز)، وهو مبلغ مثالي لمجموعة واسعة منتطبيقات البروتين، بما في ذلك تحليل مؤشر أسعار المنتجين من قبل تقارب مطياف الكتلة تنقية (أب-مس). نحن تصف تصاميمنا ل 1 L و 5 L أقفاص لمجموعات الجنين التي يمكن أن تكون بسهولة وتكلفة اقامة في أي مختبر. ونحن نقدم أيضا بروتوكول عام لجمع الجنين واستخراج البروتين لتوليد ليسيتس التي يمكن استخدامها مباشرة في التطبيقات المصب مثل أب-مس. هدفنا هو توفير وسيلة متاحة لجميع الباحثين لتنفيذ تحليلات مؤشر أسعار المنتجين في الجسم الحي .

لتوضيح استخدام هذا البروتوكول في تجربة تنقية معقدة البروتين، أنشأنا خط متماثل قابلة للحياة ذبابة، الذراع إغف- إرك، معربا عن إغف الموسومة ذبابة الفاكهة خارج الخلية ينظم إشارة كيناز (إرك، المشفرة من قبل الجينات توالت ) ت?...

Watch this Scientific Journal Video about Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments at JoVE.com

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

الهدف من هذا البروتوكول هو توفير نهج مباشر وغير مكلفة لجمع الأجنة ذبابة الفاكهة على نطاق متوسط ​​(0.5-1 ز) وإعداد مقتطفات البروتين التي يمكن استخدامها في التطبيقات البروتين المصب، مثل تقارب مطياف الكتلة تنقية (أب-مس ).

إعداد على نطاق متوسط ​​من<em&gt; ذبابة الفاكهة</em&gt; مقتطفات الجنين للتجارب بروتيوميك

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

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Research

JoVE Journal

Developmental Biology

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

7.8K Views.  University of Massachusetts Boston.  الهدف من هذا البروتوكول هو توفير نهج مباشر وغير مكلفة لجمع الأجنة ذبابة الفاكهة على نطاق متوسط ​​(0.5-1 ز) وإعداد مقتطفات البروتين التي يمكن استخدامها في التطبيقات البروتين المصب، مثل تقارب مطياف الكتلة تنقية (أب-مس ). 

Video: Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Microbead زرع الأجنة الزرد

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. 
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

إعداد الميتوكوندريا المخصب الكسور لتحليل التمثيل الغذائي في<em&gt; ذبابة الفاكهة</em

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

التصوير الهياكل التحت خلوية في الأجنة الحية اسماك الزرد

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

فؤاد وميكرورهيولوجي في الخلية صفار الجنين الزرد

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on fish abdomens to expel the eggs. An alternative method uses the pressure of gas injected into the body cavity, which causes the subsequent release of the eggs. This method is believed to have less negative effects on both the welfare and egg quality of the broodstocks. Herein, we compare the results of air and hand stripping methods with respect to one-year survival and egg quantity and quality in two salmonid fish, rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta morpha fario). Our results indicate that air stripping yielded a better quality of eggs and higher one-year survival rate in rainbow trout. In addition, air stripping resulted in lower mortality rate than the group subjected to hand stripping (25% vs. 35%). The pH and hatching rate of the hand stripped group was lower than those of the air stripped group. In the case of brown trout, the quality of eggs obtained by both hand and air-stripping methods was similar; however, the one-year losses in fish were higher in air stripped group (15% compared to 0% in hand stripped fish). Although the advantages of air stripping method over hand stripping in terms of egg quality might not be observed in all salmonid species, the air-stripping procedure might be a promising option to be adopted in hatcheries as it ensures a high level of reproducibility and efficiency.

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario

The principal aim of this study is to standardize and test the pneumatic method (air stripping) of collecting eggs in rainbow trout and brown trout. This method allows effective and simple collection of the eggs without the necessity of fish abdomen massage.

فعالية "تجريد الهواء" في "اثنين من أسماك السلمون"، تراوت قوس قزح (Oncorhynchus Mykiss) وسمك السلمون المرقط البنى (Salmo Trutta مورفى فريو)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on ...

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal metabolism. By combining the vast array of Drosophila genetic tools with the ability to survey large swaths of intermediary metabolism, a metabolomics approach can reveal complex interactions between diet, genotype, life-history events, and environmental cues. In addition, metabolomics studies can discover novel enzymatic mechanisms and uncover previously unknown connections between seemingly disparate metabolic pathways. In order to facilitate more widespread use of this technology among the Drosophila community, here we provide a detailed protocol that describes how to prepare Drosophila larval samples for gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis. Our protocol includes descriptions of larval sample collection, metabolite extraction, chemical derivatization, and GC-MS analysis. Successful completion of this protocol will allow users to measure the relative abundance of small polar metabolites, including amino acids, sugars, and organic acids involved in glycolysis and the TCA cycles.

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

This protocol describes how to prepare Drosophila larvae for GC-MS-based metabolomic analysis.

إعداد عينات اليرقات المورفولوجية للفصل اللوني للغاز-الكتلي (GC-MS)-على أساس جميع

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal ...

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

ميكانيكا خلية السبر مع ملاقط خالية من حبة الضوئية في الجنين المورفولوجية

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

نقل الاجنه طفيف التوغل والتزجيج الجنيني في مرحله الجنين الأمثل في نموذج الأرنب

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

التصوير الحي وتحليل تقلصات العضلات في جنين دروسوفيلا

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

ذوبان الجليد واستزراع كريوبريسيرفينج خطوط الخلايا المورفولوجية

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

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