تم إجراء الرعاية واستخدام الأسماك ولدت لنتائج ممثلة الموصوفة في هذا البروتوكول بما يتفق تماما مع المبادئ التوجيهية التي وضعتها لجنة رعاية واستخدام الحيوان المؤسسي في مستشفى بوسطن للأطفال، بروتوكول # 14-05-2673R.

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+complex+tissue+regeneration.">The zebrafish as a model for complex tissue regeneration.</a> Trends in genetics TIG. 29, 611-620 (2013).</li><li>Santoriello, C., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooked!+modeling+human+disease+in+zebrafish.">Hooked! modeling human disease in zebrafish.</a> Journal of Clinical Investigation. 122, 2337-2343 (2012).</li><li>Selderslaghs, I. W. T., Blust, R., Witters, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+study+of+the+zebrafish+assay+as+an+alternative+method+to+screen+for+developmental+toxicity+and+embryotoxicity+using+a+training+set+of+27+compounds.">Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity and embryotoxicity using a training set of 27 compounds.</a> Reproductive Toxicology. 33 (2), 142-154 (2012).</li><li>Lawrence, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+husbandry+of+zebrafish+(Danio+rerio):+A+review.">The husbandry of zebrafish (Danio rerio): A review.</a> Aquaculture. 269, 1-20 (2007).</li><li>Harper, C., Lawrence, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Laboratory Zebrafish (Laboratory Animal Pocket Reference). , (2010).</li><li>Nusslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish, A Practical Approach. , (2002).</li><li>Westerfield, 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> The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish. , (2007).</li><li>Carvalho, P., Arau, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+zebrafish+(Danio+rerio)+larvae+without+live+food:+evaluation+of+a+commercial,+a+practical+and+a+purified+starter+diet+on+larval+performance.">Rearing zebrafish (Danio rerio) larvae without live food: evaluation of a commercial, a practical and a purified starter diet on larval performance.</a> Aquaculture Research. 37, 1107-1111 (2006).</li><li>Spence, R., Gerlach, G., Lawrence, C., Smith, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+and+ecology+of+the+zebrafish,+Danio+rerio.">The behaviour and ecology of the zebrafish, Danio rerio.</a> Biol Rev Camb Philos Soc. 83 (1), 13-34 (2008).</li><li>Best, J., Adatto, I., Cockington, J., James, A., Lawrence, C. <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+method+for+rearing+first-feeding+larval+zebrafish:+polyculture+with+Type+L+saltwater+rotifers+(Brachionus+plicatilis).">A novel method for rearing first-feeding larval zebrafish: polyculture with Type L saltwater rotifers (Brachionus plicatilis).</a> Zebrafish. 7 (3), 289-295 (2010).</li><li>Lawrence, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+zebrafish+husbandry+and+management.">Advances in zebrafish husbandry and management.</a> Methods in Cell Biology. 104, 429-451 (2011).</li><li>Lawrence, C., Sanders, E., Henry, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+culturing+saltwater+rotifers+(Brachionus+plicatilis)+for+rearing+larval+zebrafish.">Methods for culturing saltwater rotifers (Brachionus plicatilis) for rearing larval zebrafish.</a> Zebrafish. 9, 140-146 (2012).</li><li>Tucker, C. S., Hargreaves, J. 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> Biology and Culture of Channel Catfish. 34, 634-657 (2004).</li></ol>

يعمل EC هنري ريد تربية الأحياء البحرية، وشركة، وهي الشركة التي توفر الدوارات ومركزات الطحالب، وغيرها من الإمدادات لتربية الأحياء المائية والأسماك أسواق الهاوي.

التنفيذ الناجح لأسلوب متعدد الأنواع حيدة الخلية والروتيفر لتغذية أوائل الزرد اليرقات يتطلب بروتوكولات فعالة لمهمتين: إنشاء وصيانة نظام الثقافة حيدة الخلية والروتيفر المستمر لتغذية الأسماك، وزراعة تغذية أول اليرقات الزرد جنبا إلى جنب مع الدوارات في الدبابة نفسها. ...

الزرد (دانيو rerio) هو حيوان مختبر البارز تستخدم في عدد متزايد من التخصصات العلمية، بما في ذلك ولكن لا تقتصر على الوراثة التنموية، وعلم السموم، والسلوك وتربية الأحياء المائية، البيولوجيا التجديدي، ونمذجة العديد من الاضطرابات الإنسان 1-5. على الرغم من أن الأنواع من السهل نسبيا للحفاظ في المختبر، وهناك عدد من التحديات الإدارية المرتبطة ثقافتهم 6. وأبرز هذه هي تربية اليرقات، خاصة عندما الأسماك لأول مرة تبدأ في وقت لاحق لتغذية التضخم المثانة الغاز 7. تحت الظروف العادية، التي تسيطر عليها، وهذا الحدث التنموي يحدث في ~ 5 أيام بعد الإخصاب (إدارة الشرطة الاتحادية)، مع 3 التالية - 5 أيام من النمو يجري حاسما بشكل خاص 7. الصعوبات الفنية المركزية خلال هذه المرحلة في الاستجابة على نحو كاف لمطالب الغذائية من اليرقات التغذية الأولى - يجب أن البنود تغذية تكون بحجم مناسب، digestible، جذابة، ومتاحة على أساس مستمر تقريبا، دون خلق النفايات المفرطة في خزانات زراعة. تاريخيا قد تحقق هذا عادة من خلال تقديم العديد من كميات صغيرة من الأعلاف للأسماك في صهاريج، جنبا إلى جنب مع روتين 8،9 صرف المياه. في حين أن هذه الأساليب هي إلى حد ناجحة، فهي غير فعالة، تتطلب مدخلات العمل عالية، والعودة المتغير الوحيد ومعدلات محدودة للنمو والبقاء على قيد الحياة 10. في الطبيعة، اليرقات الزرد إطعام يفترض على وفرة صغيرة الحاضر العوالق الحيوانية في عمود الماء 11. لهذا السبب، بروتوكولات تربية اليرقات التي تتضمن لقطات حية مثل المتناعلة، الدوارات، والأرتيميا وعادة ما تكون الأكثر فعالية 7. في عام 2010، أظهر أفضل والمشاركين فيها أنه كان من الممكن أن تنمو اليرقات الزرد في ثابت، والمياه المالحة مع المياه المالحة الدوارات لمدة 5 أيام الأولى من التغذية الخارجية (12). هذا النهج الذي تسخيروفاق إنتاجية عالية الطبيعية الثقافات حيدة الخلية والروتيفر لتوفير وافرة، فريسة ذات قيمة غذائية عالية دون تلويث المياه، والمحاصيل معدلات عالية جدا من نمو اليرقات والبقاء على قيد الحياة مع مدخلات العمل منخفضة 12،13. في السنوات الأخيرة، تبنت عددا متزايدا من المختبرات حول العالم أشكال مختلفة من هذا البروتوكول، والعديد من زراعة الآن الدوارات بطريقة مستمرة لدعم نظم الحضانة 14. على مدى السنوات القليلة الماضية، تم تنقيح طرق لكلا حيدة الخلية والروتيفر / متعدد الأنواع الزرد والإنتاج حيدة الخلية والروتيفر وتحسينها لتصبح أكثر توحيدا وقابلة بسهولة. توفر هذه المقالة خطوة بخطوة تعليمات ل1) إنتاج حيدة الخلية والروتيفر المستمر والقوي و2) إنشاء حيدة الخلية والروتيفر / الزرد نظام متعدد الأنواع المستخدمة لدعم النمو القوي من الأسماك لمدة 5 أيام الأولى من التغذية الخارجية.

<table border="0" cellpadding="0" cellspacing="0" width="823">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Rotifer Culture Infrastructure</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">100 Liter Culture Vessel</td>
			<td style="width:135px;">Aquaneering</td>
			<td style="width:119px;">Custom</td>
			<td style="width:264px;">Polycarbonate culture vessel, conical bottomed, with drain valve</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">5 Gallon Culture Bucket Kit</td>
			<td style="width:135px;">Reed Mariculture</td>
			<td style="width:119px;">CCS Starter Kit</td>
			<td style="width:264px;">Small volume culture vessel for small facilities</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Rigid Clear Tubing 1/2&#34; O.D., 36&#8221;</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">16025</td>
			<td style="width:264px;">Rigid clear tubing for air delivery</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Mesh tube</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">RT444X</td>
			<td style="width:264px;">Mesh tube support for floss filter</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Rotifer Floss</td>
			<td style="width:135px;">Reed Mariculture</td>
			<td style="width:119px;">Rotifer floss 12&#8221; x 42&#8221;</td>
			<td style="width:264px;">Particulate waste trap</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Peristaltic Metering Timer Pump, 5 GPD</td>
			<td style="width:135px;">Grainger</td>
			<td style="width:119px;">38M003&#160;</td>
			<td style="width:264px;">Metering pump with timer for dosing feed to rotifers</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Peristaltic Metering Timer Pump, 1-100 mL/h (for smaller-scale culture)</td>
			<td style="width:135px;">Coral Vue</td>
			<td style="width:119px;">SKU: IC-LQD-DSR</td>
			<td style="width:264px;">Metering pump with timer for dosing feed to rotifers</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Silicone Tubing&#160;</td>
			<td style="width:135px;">Cole Parmer</td>
			<td style="width:119px;"></td>
			<td style="width:264px;">Tubing for algae delivery to rotifer vessel</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Rigid Clear Tubing &#34; O.D.,36&#8221;</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">16025</td>
			<td style="width:264px;">Rigid clear tubing for air delivery to algae paste</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Rigid Clear Tubing O.D., 36&#8221;</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">16025</td>
			<td style="width:264px;">Rigid clear tubing for algae delivery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Rotifers</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Live Rotifers Brachionus plicatilis Type L</td>
			<td style="width:135px;">Reed Mariculture</td>
			<td style="width:119px;">Type L 5 million</td>
			<td style="width:264px;">Rotifer stock culture for system startup</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Rotifer Feed</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:305px;">Sodium hydroxymethylsulfonate</td>
			<td style="width:135px;">Reed Mariculture</td>
			<td style="width:119px;">ClorAm-X&#174; 1lb tub</td>
			<td style="width:264px;">Ammonia reducer for algae feed mix</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Sodium Bicarbonate</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">S25533B</td>
			<td style="width:264px;">pH buffer for algae feed mix</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:305px;">Microalgae concentrate</td>
			<td style="width:135px;">Reed Mariculture</td>
			<td style="width:119px;">Rotigrow Plus&#174; 1 liter bag</td>
			<td style="width:264px;">Nutritionally optimized rotifer feed</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Water Preparation</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="96">
			<td height="96" style="height:96px;width:305px;">&#160;Reef Crystals Reef Salt</td>
			<td style="width:135px;">That Fish Place</td>
			<td style="width:119px;">198210</td>
			<td style="width:264px;">Salt for making culture water (NOTE: this item is an example only; any contaminant free salt formulations may be used).&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Refractometer</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">SR6</td>
			<td style="width:264px;">measuring salinity</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Rotifer Culture Equipment</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Plankton Collectors 12&#34; Dia, 53 microns</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">BBPC20</td>
			<td style="width:264px;">Mesh screen for collecting rotifers</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Scrub Pads</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">SCR-58</td>
			<td style="width:264px;">Scrub pad for cleaning inside of culturing vessels</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Scrub Brush</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Bucket</td>
			<td style="width:135px;">Grainger Supply&#160;</td>
			<td style="width:119px;">43Y530</td>
			<td style="width:264px;">Graduated bucket for mixing culture water</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Hatching Jar</td>
			<td style="width:135px;">Pentair Aquatic Ecosystems</td>
			<td style="width:119px;">J30</td>
			<td style="width:264px;">Storage of algae feed mix</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Lugol&#8217;s Solution, Dilute</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">S99481</td>
			<td style="width:264px;">Agent used to immobilize live rotifers for counting</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Sedgewick-Rafter plankton counting slide with grid&#160;</td>
			<td style="width:135px;">Pentair Aquatic Eco-Systems</td>
			<td style="width:119px;">M415</td>
			<td style="width:264px;">Counting rotifers</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Miscelleneous</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:305px;">Tea Strainer</td>
			<td style="width:135px;">Kitchenworks</td>
			<td style="width:119px;">971972</td>
			<td style="width:264px;">Used for collecting zebrafish embryos after spawning</td>
		</tr>
	</tbody>
</table>

the complete and updated "rotifer polyculture method" for rearing first feeding zebrafish

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science. One of the most demanding technical aspects of culture of this species in the laboratory is rearing first-feeding larvae to the juvenile stage with high rates of growth and survival. The central management challenge of this developmental period revolves around delivering highly nutritious feed items to the fish on a nearly continuous basis without compromising water quality. Because larval zebrafish are well-adapted to feed on small zooplankton in the water column, live prey items such as brachionid rotifers, Artemia, and Paramecium are widely recognized as the feeds of choice, at least until the fish reach the juvenile stage and are able to efficiently feed on processed diets. This protocol describes a method whereby newly hatched zebrafish larvae are cultured together with live saltwater rotifers (Brachionus plicatilis) in the same system. This polyculture approach provides fish with an "on-demand", nutrient-rich live food source without producing chemical waste at levels that would otherwise limit performance. Importantly, because the system harnesses both the natural high productivity of the rotifers and the behavioral preferences of the fish, the labor involved with maintenance is low. The following protocol details an updated, step-by-step procedure that incorporates rotifer production (scalable to any desired level) for use in a polyculture of zebrafish larvae and rotifers that promotes maximal performance during the first 5 days of exogenous feeding.

النظام الثقافة حيدة الخلية والروتيفر المستمر الموصوفة هنا هو دينامية، وأنه من الطبيعي لأعداد حيدة الخلية والروتيفر لتتقلب إلى حد صغير مع مرور الوقت إذا كان هناك اختلافات في معدلات التغذية والحصاد اليومي. سكان الدوارات في واحدة من الثقافات العاملة...

Watch this Scientific Journal Video about The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish at JoVE.com

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This "polyculture" strategy promotes high growth and survival with minimal labor and disturbance to the larvae.

و&quot;حيدة الخلية والروتيفر طريقة الاستزراع متعدد الأنواع&quot; كاملة ومحدثة للتربية اسماك الزرد الأولى تغذية

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science.  One of the most demanding technical aspects of ...

the-complete-updated-rotifer-polyculture-method-for-rearing-first

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

15.3K Views.  Boston Children's Hospital. Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This "polyculture" strategy promotes high growth and survival with minimal labor and disturbance to the larvae.

Video: The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

تصوير لايف الفطرية المناعي والتفاعلات سابق للورم الخلية باستخدام نظام التعبير / UAS محرض GAL4 في اليرقات الزرد الجلد

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

استخدام نظام تيتون لدراسة الآليات الجزيئية لتجديد الزرد

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

تحليل الزرد اليرقات الهيكل العظمي النزاهة العضلات مع ايفانز الأزرق صبغ

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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 ...

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

تربية ذبابة الفاكهة<em&gt; ذبابة الفاكهة</em&gt; تحت ممحوضة ومعروف المعايشات شروط

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

حمل كاملة ورم التجديد من مجافي الفم المنفخة من البحر شقائق النعمان النجيم<em&gt; Nematostella vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

في المختبر نمو بصيلات بريانترال الماوس تحت ظروف محاكاة الجاذبية الصغرية

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

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. ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

التصور للنشاط الكهربائي الخلوية في الزرد الأجنة في وقت مبكر، والأورام

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

أسلوب في فيفو لدراسة سلامة حاجز الدم-الخصية الماوس

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

و"حيدة الخلية والروتيفر طريقة الاستزراع متعدد الأنواع" كاملة ومحدثة للتربية اسماك الزرد الأولى تغذية

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