ويدعم أبحاثنا المختبر من قبل برنامج بحوث جماعية من المعاهد الوطنية للصحة ، المعهد الوطني لتعاطي الكحول والإدمان على الكحول.

<ol>
	<li>Eccles, J. 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+action+potential+of+the+superior+cervical+ganglion.">The action potential of the superior cervical ganglion.</a> J Physiol. 85, 179-206 (1935).</li><li>Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-dependent+modulation+of+N-type+calcium+channels+by+G-protein+&#x03B2;&#x03B3;+subunits.">Voltage-dependent modulation of N-type calcium channels by G-protein &#x03B2;&#x03B3; subunits.</a> Nature. 380, 255-258 (1996).</li><li>Schofield, G. G., Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+currents+of+acutely+isolated+adult+rat+superior+cervical+ganglion+neurons.">Potassium currents of acutely isolated adult rat superior cervical ganglion neurons.</a> Brain Res. 485, 205-214 (1989).</li><li>Schofield, G. G., Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+and+calcium+currents+of+acutely+isolated+adult+rat+superior+cervical+ganglion+neurons.">Sodium and calcium currents of acutely isolated adult rat superior cervical ganglion neurons.</a> Pflugers Arch. 411, 481-490 (1988).</li><li>Dean, D. A., Goldman, R. D., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4.">Chapter 4.</a> Live Cell Imaging: a Laboratory Manual. 1, 51-66 (2005).</li><li>Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+expression+of+receptors+and+signaling+proteins+in+adult+mammalian+sympathetic+neurons+by+microinjection.">Heterologous expression of receptors and signaling proteins in adult mammalian sympathetic neurons by microinjection.</a> Methods Mol Biol. 83, 191-202 (1997).</li><li>Ikeda, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+G-protein+signaling+components+in+adult+mammalian+neurons+by+microinjection.">Expression of G-protein signaling components in adult mammalian neurons by microinjection.</a> Methods Mol Biol. 259, 167-181 (2004).</li><li>Ikeda, S. R., Jeong, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+RGS-insensitive+G&#x03B1;+subunits+to+study+endogenous+RGS+protein+action+on+G-protein+modulation+of+N-type+calcium+channels+in+sympathetic+neurons..">Use of RGS-insensitive G&#x03B1; subunits to study endogenous RGS protein action on G-protein modulation of N-type calcium channels in sympathetic neurons..</a> Methods Enzymol. 389, 170-189 (2004).</li><li>Washbourne, P., McAllister, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+gene+transfer+into+neurons.">Techniques for gene transfer into neurons.</a> Curr Opin Neurobiol. 12, 566-573 (2002).</li><li>Zhang, Y., Yu, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+microinjection+technology+in+cell+biology.">Single-cell microinjection technology in cell biology.</a> Bioessays. 30, 606-610 (2008).</li><li>Vivaudou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+G-protein+regulation+of+GIRK1+and+GIRK4,+the+two+subunits+of+the+KACh+channel,+using+functional+homomeric+mutants.">Probing the G-protein regulation of GIRK1 and GIRK4, the two subunits of the KACh channel, using functional homomeric mutants.</a> J Biol Chem. 272, 31553-31560 (1997).</li></ol>

واجه المشاكل المشتركة والاقتراحات : كما ذكر ، في وقت سابق حقن النووية الناجحة تعتمد على وجود خلايا لوحات ملتصقة زراعة الأنسجة. تأجيل بدء حقن بضع ساعات بعد إجراء تفارق الخلايا العصبية يسمح للانضمام إلى أسفل من الأطباق. بالإضافة إلى ذلك ، الأطباق طلاء بنسبة 0.1 ملغ / مل من الوزن الجزيئي عالية بولي - L - يسين (سيغما ، وسانت لويس ، MO) الإيدز التصاق الخلايا العصبية على السطح السفلي من الأطباق. يمكن انسداد pipets microinjection ، وخصوصا عندما تحاول حقن تركيز عالية من الحمض النووي ، ومشكلة متكررة. يمكن استخدام أعمدة عالية الجودة لفصل وتنقية عزل DNA البلازميد ، وتنفيذ الخطوات الإضافية المذكورة تنقية (فلاتر تدور ، والغزل في أنابيب الهيماتوكريت) يساعد على إزالة الجسيمات قبل عن طريق الحقن. خلال الحقن ، ويمكن استخدام &quot;نظيف&quot; وظيفة من حاقن الضغط (ليالي 0.2 كافية) ، ولكن إذا كان هذا لا يحل المشكلة ، يجب استخدام الماصة حقن جديدة. إذا كانت غالبية pipets microinjection انسداد أثناء جلسة الحقن ، والنظر في تغيير إعدادات الزجاج مجتذب الماصة لإنتاج نصائح الماصة microinjection مع افتتاح أكبر. والمشكلة الأخرى التي تنشأ خلال الحقن النووية هو التفاوت من السطح السفلي من الأطباق زراعة الأنسجة. هذا التغير يؤثر بشكل مباشر على دقة استهداف نصائح الماصة للحقن نواة منذ عمق ثابت يخترق الماصة خلال الحقن. لذا ، لا بد من تعديل هذا الحد ض محور أثناء الحقن داخل الطبق نفسه. وسوف يكون واضحا عند إجراء تعديل ضروري : لن يكون هناك أي مؤشر على حقن النووي (أي سحابة بيضاء في نواة أو غاب كليا الخلية) ، أو طرف الماصة حقن يقترب من قاع الطبق (ضغط الخلية تحطمها أو حتى التلميح الماصة في قاع الطبق). ويمكن استخدام الزجاج استنساخ الاسطوانات (OD 10 ملم ، القط رقم 2090-01010 ، Bellco زجاج Vineland ، ونيو جيرسي.) خلال طلاء الخلايا العصبية لحصرها في منطقة أصغر حجما وأكثر محصورة ؛ بالتالي تقليل المسافة المطلوبة للتحرك والماصة بين الحقن الحقن. تتم إزالة هذه الاسطوانات الاستنساخ قبل تنفيذ microinjections. عيوب التقنية : microinjections Intranuclear يطالبون تقنيا ، والتي تتطلب الصبر وعلى درجة عالية من البراعة اليدوية من قبل المشغل. الكفاءة مع هذه التقنية ، كما هو الحال مع أي مهارة أخرى ، ويأتي مع الممارسة. وهكذا ، ينصح لممارسة حقنا النووي من الجينات وحدها قبل محاولة مراسل التجارب الفعلية. لمزيد من المساعدة في عملية الحقن ، قد يكون من الممكن لترسيخ خطوات micromanipulator حاقن والضغط في برنامج الكمبيوتر. ويمكن استخدام برامج مثل ايغور برو (WaveMetrics ، بحيرة أوسويجو ، OR) لتخزين الإعدادات microinjection وبرنامج التحكم متعددة الوظائف (ShuttlePro ، كونتور تصميم ، ويندهام ، NH) إلى شبه أتمتة عملية الحقن (انظر ل6،7،8 مزيد من التفاصيل). عيب آخر من تقنية microinjection intranuclear هي نسبة النجاح منخفضة. حوالي 10-20 ٪ من عمليات الحقن النووية حاول التعبير تؤدي إلى البروتين. هذه الكفاءة في حين قد تكون كافية للتطبيقات التي لا تحتاج إلى عدد كبير من الخلايا بالإعراب ، الكهربية على سبيل المثال ، لفحوصات البيوكيميائية المختلفة هذا قد لا يكون كافيا. تطبيقات التقنية : على الرغم من عيوبه ، microinjection intranuclear من الحمض النووي هو تقنية مفيدة للغاية للتعبير عن heterologously البروتينات في الخلايا العصبية. وحققت مستويات عالية من بروتين تعبير مع microinjections النووية نظرا لوجود عدد كبير من البلازميدات تطلق في نواة خلال الحقن ، داعية نشطة للغاية CMV تنظيم النسخ الجيني ، والاستقرار طويل من البلازميدات في النواة. أكثر من التعبير عن بروتين مفيد خصوصا في التجارب السلبية السائدة التي تتطلب مستويات عالية من البروتينات مغاير لتطغى على البروتين الذاتية. ميزة أخرى لحقن intranuclear هو القدرة على تحقيق نسبة ثابت من ثوابت [كدنا] إلى النواة عندما يتم حقن بنيات متعددة. مع أساليب أخرى ترنسفكأيشن ، فمن الصعب أن أعرض بتكاثر نفس النسبة من كل بناء الحمض النووي في خلايا مختلفة في نفس الطبق وبين transfections. الحقن المباشر للحل [كدنا] في نواة يلغي هذا المصدر من مصادر التباين ويسمح titered نسبة من البروتينات وأعرب على نحو فعال. الأساليب التقليدية ترنسفكأيشن تكون ذات فعالية محدودة في مرحلة ما بعد الإنقسامية الخلايا لأن التأسيس منخفضة من الحمض النووي في نواة (9) وسمية المواد الكيميائية المرتبطة reag ترنسفكأيشنالوالدان 10. microinjections النووية في التغلب على هذه القضايا من خلال إدخال المادة الوراثية في نواة ميكانيكيا. على الرغم من أن هذا الأسلوب هو أكثر انخراطا ، والقدرة على دراسة تأثير وجود البروتين في النظام البيولوجي الوظيفي ، مع استكمال الذاتية مسارات إشارة ، فإن أكثر من يعوض عن طبيعة شاقة من هذه التقنية. تم تنفيذ جميع الإجراءات التجريبية على الحيوانات وفقا للمعاهد الوطنية للصحة المبادئ التوجيهية لرعاية الحيوان والاستخدام.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ultrafree-MC Filter Unit</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC30VV00</td>
<td>Durapore PVDF filter; 0.1 &mu;m pore-size</td>
</tr>
<tr>
<td>TE buffer</td>
<td>&nbsp;</td>
<td>Quality Biological, Inc</td>
<td>351-010-131</td>
<td>10 mM Tris, 1 mM EDTA, pH 8.0</td>
</tr>
<tr>
<td>Micro-hematocrit capillary tubes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>22-362-574</td>
<td>Unheparinized Preparation: 
Soak in 100% ethanol overnight. Wash thoroughly with deionized water three times and dry bake glass in an oven (95&deg;C). Cut the cleaned hematocrit tubes into 3-4 cm length pieces using a diamond-tipped scriber, and seal one end closed with a flame and forceps to hold the glass. Store in a clean glass beaker and cover to prevent dust build-up. </td>
</tr>
<tr>
<td>Microcentrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5417 R</td>
<td>With a fixed angle or swinging-bucket rotor</td>
</tr>
<tr>
<td>Microloader 20 &mu;l pipet tips</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5242 956.003</td>
<td>&nbsp;</td>
<tr>
<td>Microinjection capillary glass</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW120F-4</td>
<td>Thin-walled with filament, 1.2 mm outer-diameter, 0.9 mm inner-diameter, 100 mm length</td>
</tr>
<tr>
<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument Co.</td>
<td>P-97</td>
<td>With platinum-iridium box filament (part # FB330B)</td>
</tr>
<tr>
<td>Nikon Diaphot TMD Inverted Microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>20x and 40x phase-contrast objective lenses, mounted on a vibration isolation table</td>
</tr>
<tr>
<td>Charge-coupled device (CCD) camera </td>
<td>&nbsp;</td>
<td>Cohu Inc.</td>
<td>6410</td>
<td>&nbsp;</td>
<tr>
<td>Video monitor</td>
<td>&nbsp;</td>
<td>Sony Corporation</td>
<td>PVM-122</td>
<td>Black and White, 9&#x201D; screen </td>
</tr>
<tr>
<td>Micromanipulator system</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5171</td>
<td>&nbsp;</td>
<tr>
<td>Microinjection Unit</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>FemtoJet Express</td>
<td>&nbsp;</td>
<tr>
<td>Microinjection controller</td>
<td>&nbsp;</td>
<td>Contour Design</td>
<td>S-PROV2</td>
<td>Programmable multimedia controller</td>
</tr>
<tr>
<td>Igor Pro</td>
<td>&nbsp;</td>
<td>WaveMetrics</td>
<td>Version 4.05A</td>
<td>Command program to control the microinjection controller written by S. Ikeda</td>
</tr>		</tbody>
		</table>
		</div>

intranuclear microinjection of dna into dissociated adult mammalian neurons

الخلية العصبية الأولية الثقافات هي أدوات قيمة لدراسة وظيفة البروتين لأنها تمثل نظاما بيولوجيا أكثر أهمية بالمقارنة مع خطوط الخلايا خلده. ومع ذلك ، وطبيعة مرحلة ما بعد الإنقسامية من الخلايا العصبية الأولية يمنع فعالية بروتين تعبير مغاير باستخدام إجراءات مشتركة مثل electroporation ترنسفكأيشن كيميائيا أو بوساطة. وبالتالي ، يجب أن يوظف تقنيات أخرى من أجل التعبير عن البروتينات بشكل فعال في هذه الخلايا غير الانقسام. في هذه المقالة ، نحن تصف الخطوات المطلوبة لتنفيذ عمليات الحقن intranuclear من يبني [كدنا] في فصل الخلايا العصبية المتعاطفة الكبار. يمكن لهذا الأسلوب ، الذي تم تطبيقه على أنواع مختلفة من الخلايا العصبية ، وتحدث بنجاح غيروي بروتين تعبير. المعدات الأساسية لهذا الإجراء يشمل microinjection مجهر مقلوب لتصور خلايا ، والماصة حقن زجاجية مملوءة بمحلول [كدنا] متصلة (أ) 2 N (ز) الضغط نظام التسليم ، وmicromanipulator ملف. وينسق الحركة micromanipulator حقن الماصة microinjection مع نبضة قصيرة من الضغط N 2 إلى إخراج حل [كدنا] من طرف الماصة. هذا الأسلوب لا تملك سمية المرتبطة العديد من الأساليب الأخرى ويتيح ترنسفكأيشن بنيات متعددة لتكون الحمض النووي التي أعرب عنها في نسبة ثابتة. انخفاض عدد خلايا حقن يجعل الإجراء microinjection مناسبة تماما للدراسات خلية واحدة مثل التسجيلات الكهربية والتصوير الضوئي ، ولكن قد لا تكون مثالية لفحوصات البيوكيميائية التي تتطلب عددا أكبر من الخلايا وكفاءة أعلى ترنسفكأيشن. على الرغم من microinjections intranuclear تتطلب استثمارا في المعدات والوقت ، والقدرة على تحقيق مستويات عالية من بروتين تعبير مغاير في بيئة ذات الصلة من الناحية الفسيولوجية يجعل هذه التقنية أداة مفيدة جدا للتحقيق في وظيفة البروتين.

Watch this Scientific Journal Video about Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons at JoVE.com

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

الحقن المباشر للintranuclear [كدنا] هو أسلوب فعال لترنسفكأيشن بعد الإنقسامية الخلايا. هذا الأسلوب يوفر مستويات عالية من بروتين تعبير مغاير من يبني [كدنا] واحدة أو متعددة ، وتمكن وظيفة البروتين لدراستها في بيئة ذات الصلة من الناحية الفسيولوجية مع مجموعة متنوعة من المقايسات خلية واحدة.

Intranuclear Microinjection من الحمض النووي في الخلايا العصبية فصل الثدييات الكبار

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

intranuclear-microinjection-dna-into-dissociated-adult-mammalian

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JoVE Journal

Biology

16.8K Views.  National Institutes of Health (NIH). الحقن المباشر للintranuclear [كدنا] هو أسلوب فعال لترنسفكأيشن بعد الإنقسامية الخلايا. هذا الأسلوب يوفر مستويات عالية من بروتين تعبير مغاير من يبني [كدنا] واحدة أو متعددة ، وتمكن وظيفة البروتين لدراستها في بيئة ذات الصلة من الناحية الفسيولوجية مع مجموعة متنوعة من المق?...

Video: Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

التحضير للثقافات ماوس العصبية القشرية نأت

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

ترنسفكأيشن الحمض النووي لعضلات الهيكل العظمي باستخدام الثدييات في فيفو Electroporation

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

تحليل مزدوج الجديلة إصلاح الحمض النووي (DSB) فاصل في خلايا الثدييات

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

حقن داخل الصفاق الى اسماك الزرد الكبار

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

تحليل حركية مرنا الصادرات النووية في خلايا الثدييات التي Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

تحليل وظيفة GTPases الصغيرة التي Microinjection من البلازميدات إلى الخلايا الظهارية المستقطب

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

زرع الخلايا مباشرة في الكلى من اسماك الزرد الكبار

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

في الجسم الحي من Electroporation Morpholinos في شبكية العين اسماك الزرد الكبار

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

كفاءة تمايز الخلايا الجذعية الجنينية في الخلايا العصبية الحركية

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.