We are indebted to Pierre Mattar and Freda Miller for their assistance with the in utero electroporation and to Noel Ghanem and Bensun Fong for creating diagrams. We thank Linda Jui and Jason G. MacLaurin for excellent technical assistance. Equipment was supported by the Centre for Stroke Recovery.
This work was supported by a scholarship from HSFO and OGS to D.S.S. and a CIHR grant to R.S.S.

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The authors have nothing to disclose

اثنين من التحديات الرئيسية في أداء في electroporations الرحم هي 1) منع الفتك الجنين و2) تقليل التباين بين electroporations. واحدة من أهم العوامل في منع الفتك هو نوعية الإبرة، والإبر غير حادة تلحق المزيد من الضرر للجنين. عندما قطع الإبرة، والحفاظ على طرف لأطول فترة ممكنة في حي...

الهجرة الشعاعية هي عملية مركزية في التنمية القشرية في وقت مبكر. ذلك يعتمد على مجموعة متنوعة من العوامل خلية مستقلة، مثل الصحيح للخروج الإنقسامية وتمايز الخلايا العصبية، الخلايا القطبية، وتنظيم ديناميات هيكل الخلية والتعبير عن مستقبلات عبر الغشاء، فضلا عن عدم خلية العوامل المستقلة، مثل تشكيل سقالة الدبقية شعاعي و إفراز التوجيه المهاجرة جزيئات 1-3. منذ تعطل أي من هذه الآليات يمكن أن يعوق هجرة الخلايا العصبية في نموذج الفأر المعدلة وراثيا 4-8، وتحديد السبب الكامن وراء وجود خلل في الهجرة يمكن أن يكون عملية معقدة وصعبة. في الرحم Electroporation لل(IUE) يمكن استخدامها لاستكمال وتبسيط تفسير النمط الظاهري من نموذج الجيني خروج المغلوب، وبالتالي توضيح الآليات الهامة المطلوبة للهجرة شعاعي 7،9-11. في الرحم Electroporation لل(IUE) هو العملية التي من carryi بلازميد<html> نانوغرام كل من الجينات في المصالح ومراسل يتم ضخها في البطين في دماغ فأر أو جرذ الجنين ومن ثم رسمها في الخلايا المبطنة للبطين من خلال استخدام تيار كهربائي 12-14. وهذا يسمح للمحقق لتحليل تأثير أعلى أو لأسفل التنظيم من الجينات في المصالح في التنمية أو وظيفة الخلايا العصبية electroporated. وبعد IUE، والعقول يمكن معالجتها لالمناعية 7،9-11، الكهربية 15 أو خلية الثقافة 16،17. والميزة الرئيسية لIUE هي التي تسمح للتلاعب محددة للغاية في التعبير الجيني. وعلاوة على ذلك، يمكن أن تستخدم IUE لاستهداف منطقة محددة من الدماغ النامية من خلال توجه الحالي (الشكل 2). ويمكن أيضا أن تستخدم IUE لاستهداف نوع خلية معينة من خلال حقن البلازميدات تحمل الجينات تحت سيطرة المروجين مختلف أو الأنظمة تفعيل بلازميد (التتراسيكلين الناجم عن التعبير الجيني هو مثال) 10،18-21. jove_content &quot;&gt; IUE يمكن استخدامها جنبا إلى جنب مع لجنة المساواة العرقية، السلمون المدخن الجينات بوساطة نظام الختان 7-9،11،22. يمكن electroporated البلازميد التي تحتوي على الجين ترميز الرسالة للبروتين لجنة المساواة العرقية إلى متماثلة اللواقح الجنين لأليل floxed ( التي يتم يحيط المناطق الجينات أو الحمض النووي محددة من قبل اثنين من المواقع loxP لجنة المساواة العرقية بوساطة إعادة التركيب DNA). سوف لجنة المساواة العرقية ثم لحث على إعادة التركيب من الجينات في المصالح وتحديدا في السلائف العصبية electroporated، وتوليد الضربة القاضية للخروج من floxed allele.The تأثير البروتين ضربة قاضية على الهجرة العصبية والتنمية داخل الخلايا العصبية الفردية يمكن بعد ذلك درس. Electroporation للمن لجنة المساواة العرقية يدفع إعادة التركيب إلا في عدد صغير من السكان خلايا المصابة، وترك البيئة الداعمة سليمة. في المقابل، التعبير أنسجة معينة من لجنة المساواة العرقية تحت السيطرة من نوع خلية معينة المروج، يحدث في جميع أنحاء الأنسجة كامل بحيث أن كلا neuroblasts المهاجرة والبيئة المحيطة يمكن أن تتأثر. وهكذا، juxtaposition من هذين النهجين يمكن تحديد ما إذا كان هو عيب الهجرة نظرا يرجع إلى الخلية آليات غير المتمتعة بالحكم الذاتي أو الحكم الذاتي الخلية. الهجرة المعيبة في كل من النظم التجريبية تشير إلى أن النتائج النمط الظاهري المرصودة من آلية خلية مستقلة. الهجرة العادية التالية Electroporation للمن لجنة المساواة العرقية مع الهجرة المعيبة في نموذج محدد لجنة المساواة العرقية الأنسجة تشير إلى أن الجينات في المصالح يتصرف من خلال آلية غير خلية مستقلة. ويمكن أيضا أن تستخدم لIUE إجراء تجارب الانقاذ electroporating الجينات تتفاعل المحتملة في ضرب الحيوانات 7،9،10. على سبيل المثال، يمكن أن محقق محاولة لانقاذ النمط الظاهري الهجرة في نموذج المعدلة وراثيا التي كتبها electroporating هدفا المصب وتحديد ما إذا كان يتم تصحيح الخلل الهجرة في الخلايا العصبية electroporated. وهذا له فائدة إضافية أن انقاذ ناجحة يشير إلى أن الهجرة العادية يمكن استعادة عن طريق التلاعب البروتين التعبير في جمعية مهندسي البترولمحدَّدة الخلايا العصبية، على الرغم من أن البيئة المحيطة لا تزال قاصرة عن الجينات المستهدفة. مرة أخرى، وهذا النهج هو المزيد من الوقت وفعالة من حيث التكلفة من عبور أو توليد خطوط المعدلة وراثيا، مع فائدة إضافية تتمثل في تحديد ما إذا كانت آلية المعيبة هي خلية مستقلة. IUE يمكن استخدامها لتتبع هجرة الخلايا العصبية من خلال Electroporation للمن البلازميد مراسل في ضرب الأجنة (الشكل 4C). كما فقط الخلايا العصبية تبطن البطين في وقت الجراحة وelectroporated 17، IUE يمكن استخدامها لتتبع الخلايا العصبية ولدوا في وقت محدد مع الاستفادة من التصور من التشكل في الجسم الحي. وأخيرا، فمن الممكن استخدام IUE لاستهداف مناطق الدماغ مثل الدماغ الانتهائي وسطي أو الظهرية، الحصين أو البارزة العقدية (الشكل 2). وهذا يعطي الباحثين القدرة على تحقيق وظيفة الجين في منطقة مستقلة محدد من مضاعفات نتائج لجي من البروتين ضربة قاضية في الهياكل المجاورة. البروتين الشبكية (PRB)، P107 P130 وتتكون الأسرة من البروتين جيب وراسخة المنظمين للدورة الخلية الخروج. ومع ذلك، هناك أدلة متزايدة على أن هذه البروتينات أيضا تنظيم دورة الخلية جوانب مستقلة من التطور العصبي. كما بينا سابقا، وPRB P107 تلعب دورا حاسما في كل من عرضية 4،23 والهجرة شعاعي 6. هنا، علينا أن نظهر دور البروتين جيب أفراد الأسرة PRB وP107 على الهجرة العصبية 6 لتجسيد استخدام في الرحم Electroporation للمن لجنة المساواة العرقية في نموذج المعدلة وراثيا. وباختصار، IUE يوفر وسيلة قوية لتحليل الخلايا آثار الحكم الذاتي في حذف الجينات (أو overexpression). عندما جنبا إلى جنب مع أنسجة محددة ضرب النماذج، يمكن IUE تقديم معلومات إضافية بشأن آليات السيطرة على الهجرة العصبية.

<table border="0" cellpadding="0" cellspacing="0" width="538">
	<tbody>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:233px;">Table 1: Equipment and Materials</td>
			<td style="width:64px;"></td>
			<td style="width:64px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:119px;">Product Name</td>
			<td style="width:115px;">Company</td>
			<td colspan="2" style="width:128px;">Catalog Number</td>
			<td style="width:177px;">Description</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:119px;">ECM 830 Square Wave Electroporator</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0052</td>
			<td style="width:177px;">Electroporator only &#8211; buy cables separately (45-0204)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">1250FS Footswitch for ECM 830</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0211</td>
			<td style="width:177px;">Foot paddle is necessary for hands-free electroporation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Tweezertrodes</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0489 (5mm)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 45-0488 (7mm)</td>
			<td style="width:177px;">Platinum forcep style electrodes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Femtojet</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920010504</td>
			<td style="width:177px;">Microinjector</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Griphead 0</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007414</td>
			<td style="width:177px;">Holds the pulled needle</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Universal Capillary Holder</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007392</td>
			<td style="width:177px;">Connects griphead to tube</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Injection Tube</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007431</td>
			<td style="width:177px;">Connects capillary holder to Femtojet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Foot Control</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920005098</td>
			<td style="width:177px;">Foot paddle for hands-free injection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Microloader Tips</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">930001007</td>
			<td style="width:177px;">Tips for loading the pulled capillary tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Plasmid Mega Kit</td>
			<td style="width:115px;">Qiagen</td>
			<td colspan="2" style="width:128px;">12181</td>
			<td style="width:177px;">5 QIAGEN-tip 2500, Reagents, Buffers</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Fast Green FCF</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td colspan="2" style="width:128px;">F7252-5G</td>
			<td style="width:177px;">Non-toxic dye</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Extra Fine Bonn Scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">14084-08</td>
			<td style="width:177px;">For trimming the needles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Bonn Strabismus Scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">14084-09</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Graefe Forceps</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">11050-10</td>
			<td style="width:177px;">Straight</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Colibri Retractors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">17000-03</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Stapler</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">12031-07</td>
			<td style="width:177px;">For 7mm clips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Castroviejo Needle Holder</td>
			<td style="width:115px;">Medical Tools</td>
			<td colspan="2" style="width:128px;">SNH-6737</td>
			<td style="width:177px;">Straight, with lock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Needle Puller</td>
			<td style="width:115px;">Narishige</td>
			<td colspan="2" style="width:128px;">PC-10</td>
			<td style="width:177px;">For pulling microcappillaries</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Microcapillaries</td>
			<td style="width:115px;">Drummond</td>
			<td colspan="2" style="width:128px;">1-000-800</td>
			<td style="width:177px;">0.4 mm inner diameter glass capillary tubes</td>
		</tr>
	</tbody>
</table>

induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models

حذف الوراثي باستخدام نظام جنة المساواة العرقية، السلمون المدخن في خطوط الماوس المعدلة وراثيا هو أداة قوية تستخدم لدراسة وظيفة البروتين. ومع ذلك، إلا في نماذج محددة للغاية لجنة المساواة العرقية، الحذف من البروتين طوال السكان الأنسجة أو الخلايا غالبا ما يؤدي إلى الظواهر المعقدة الناجمة عن آليات التفاعل متعددة. تحديد ما إذا كانت النتائج النمط الظاهري من تعطل آلية مستقلة خلية، وهو جوهري إلى الخلية في السؤال، أو من آلية مستقلة غير الخلية، التي من شأنها أن تنجم عن ضعف البيئة تلك الخلية، ويمكن أن يكون من الصعب تمييز. للحصول على نظرة ثاقبة وظيفة البروتين في سياق في الجسم الحي، في الرحم Electroporation لل(IUE) يتيح حذف الجينات في مجموعة فرعية صغيرة من الخلايا داخل القشرة تطوير أو بعض منطقة في الدماغ مختارة أخرى. يمكن أن تستخدم IUE لاستهداف مناطق محددة في الدماغ، بما في ذلك الدماغ الانتهائي الظهرية، الدماغ الانتهائي وسطي، الحصين، أو البارزة العقدية. وهذا يسهل س الملاحظةو عواقب خلية مستقلة حذف الجينات في سياق بيئة صحية. والهدف من هذا البروتوكول هو لاظهار كيف يمكن استخدام IUE لتحليل وجود خلل في الهجرة شعاعي في خط الماوس المعدلة وراثيا floxed، مع التأكيد على التمييز بين الخلية آثار الحكم الذاتي مستقلة وغير خلية من البروتين الحذف. بمقارنة النمط الظاهري الناتج عن حذف الجينات داخل القشرة بأكملها مقابل IUE بوساطة حذف الجينات في خلية السكان محدود، مزيد من التبصر في وظيفة البروتين في نمو الدماغ ويمكن الحصول من استخدام إما تقنية في عزلة.

Different regions of the brain can be targeted for electroporation by varying the orientation of the paddle electrodes over the embryo&#8217;s head (Figure 2). For all parts of the forebrain, the plasmid is injected into the lateral ventricle (the hind brain can be targeted by injecting into the fourth ventricle). In general, place the electrodes so that the straight line between them passes through the region of interest, with DNA being drawn toward the positive electrode. To target the lateral cortex (...

Watch this Scientific Journal Video about Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models at JoVE.com

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

تحريض حذف البروتين من خلال<em&gt; في الرحم</em&gt; Electroporation لللتحديد العجز في العصبية الهجرة في النماذج المعدلة وراثيا

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

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Research

JoVE Journal

Developmental Biology

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

10.1K Views.  University of Ottawa. Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment. 

Video: Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

مقاربة Optogenetic لتقييم تشكيل الوصلات العصبية في نظام المشارك الثقافة

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

السابق الرحم Electroporation للوعضوي النمط من الثقافة شريحة ماوس الحصين الأنسجة

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

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 ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

تقنية لاستهداف حقن مكروي إلى البلدان النامية<em&gt; القيطم</em&gt; الكلى

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

وجه التفريق بين فروعه المكونة للدم البدائي والنهائي من الخلايا الجذعية Pluripotent البشرية

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

جيل من الفئران المعدلة وراثيا من خلال ميكروينجكتيون من البويضات

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

تصوير لايف من ماوس الثانوي باليت فيوجن

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

تشريح وتلطيخ إيممونوفلوريسسينت للفطر الجسم وعلم الخلايا العصبية في أدمغة البالغين melanogaster المورفولوجية

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

تطبيق [رني] والحرارة الناجمة عن صدمة "التعبير عامل النسخ" إلى "إعادة برمجة الخلايا الجرثومية" إلى الخلايا العصبية في C. ايليجانس

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

خلية "التجميع فحوصات" لتقييم ربط الشق المورفولوجية مع ترانس-يغاندس وتثبيط عن طريق رابطة الدول المستقلة-يغاندس

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...