Name: cortex-, hippocampus-, thalamus-, hypothalamus-, lateral septal nucleus- and striatum-specific in utero electroporation in the c57bl/6 mouse
Uploaded: 2016-01-18T14:00:00
Description: Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

Technical supported by Melanie Pfeifer and Nikolai Schmarowski (Institute for Microscopic Anatomy and Neurobiology, University Medical Center Mainz).

وقد أجريت التعامل مع الفئران والإجراءات التجريبية وفقا للمبادئ التوجيهية الأوروبية والوطنية والمؤسسية لرعاية الحيوان: بيان الأخلاق. 1. في الرحم Electroporation لل ملاحظة: في الرحم Electroporation ...

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This protocol describes in detail how to transfect the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus of C75BL/6 mice. With all the provided information this is the first protocol, which supplies all necessary information to easily recreate transfections of these cerebral regions in the C57BL/6 mouse. Previous publications are mostly focused only on a few specific r...

منذ أول وصف في عام 2001 من قبل ثلاث مجموعات مستقلة 1-3 ط ن الرحم Electroporation للأصبحت أداة القياسية المستخدمة على نطاق واسع لتحليل التعبير الجيني في النظام العصبي المركزي القوارض. بالمقارنة مع جيل من الفئران خروج المغلوب، الذي هو، على الرغم من تحسن مستمر التقنيات، لا يزال الوقت والمال المستهلكة، والرحم في الطعون Electroporation للبسبب بساطته. لذلك، في الرحم Electroporation للتمكن gain- بسرعة وكفاءة والخسارة من وظيفة الدراسات 4. ل transfect المناطق الدماغية، يتم حقن محلول يحتوي على البلازميد سالبة الشحنة إلى البطين. خلال النبض الكهربائي، والحمض النووي سالبة الشحنة بترحيل نحو القطب الموجب، وبالتالي يمكن تحديد المنطقة transfected ببساطة عن طريق تغيير موقف القطب الموجب. وكثيرا ما تبين أن العديد من مناطق الجهاز العصبي المركزي يمكن أن يكون تاrgeted 3،5-8. على سبيل المثال، تظهر الدراسات الحديثة transfections محدد من الحصين والقشرة الكمثرية أو المخطط 11/09. ومع ذلك، فإن المعلومات حول المواقف المناسبة غالبا ما تكون موحدة بالكاد فقط وليست دائما سهلة لنقل لسلالات مختلفة الماوس. ترنسفكأيشن بعض المراحل الجنينية لا يزال بعيدا عن تافهة. يجب أن تؤخذ العديد من العوامل التي تؤثر في الاعتبار عند اختيار مجموعة المتابعة لتحديدا في الرحم Electroporation لل. أولا، ل transfect على النحو الأمثل المراحل الجنينية منها، هناك حاجة إلى معرفة الفولتية المناسبة. الفولتية العالية تقلل من معدل البقاء على قيد الحياة، في حين الفولتية المنخفضة تقلل من 2،3،12 كفاءة ترنسفكأيشن. أيضا حجم مجداف الكهربائي يلعب دورا حاسما، لأن استخدام المجاذيف الكهربائي التي هي نتائج كبيرة جدا في خفض خصوصية أو يمكن أن يسبب الوفاة بسبب المودة من ضربات القلب 4،12،13. وتطبقالجهد وحجم وموقف مجداف الكهربائي هي أهم سمات للنظر، ولكن هناك أيضا عوامل أخرى تؤثر على نتائج Electroporation لل، مثل كمية التطبيقية من الحمض النووي الحل. قمنا بتطوير بروتوكول مفصلة والتي تمكن ترنسفكأيشن سريعة وفعالة من مختلف المناطق الدماغية من الفأرة 12 C57BL / 6. في هذا البروتوكول على معلومات مفصلة حول الفولتية لاستخدامها ويتم توفير حجم مجداف الكهربائي لتعزيز خصوصية. وعلاوة على ذلك، معلومات عن البطين المراد شغلها جنبا إلى جنب مع ويتم تزويد توصيات لكمية من محلول البلازميد وموقف القطب. في إشارة إلى معلومات الموقع مفصلة في خريطة والتصور مزيد من هذه المواقف يمكن محددة واضحة وفعالة في الرحم Electroporation للمن القشرة خلف الطحال، والقشرة الحركية، والقشرة الحسية الجسدية، والقشرة الكمثرية، رانه قرن آمون 1-3، والتلفيف المسنن، المخطط، نواة الحاجز الأفقي، والمهاد وتحت المهاد.

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:215px;">QIAGEN</td>
			<td style="width:188px;">12362</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;">Fast Green</td>
			<td>Roth</td>
			<td>0301.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCAGGS</td>
			<td>Addgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">borosilicate glass capillaries (0.8-0.9 mm diameter)</td>
			<td>Wold Precision Instrument Inc.</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane (Forene)</td>
			<td>Abbott</td>
			<td>PZN 4831850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carprofen (Rimadyl)</td>
			<td>Pfizer GmbH</td>
			<td>approval number: 400684.00.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eye ointment (Bepanthen Augen und Nasensalbe)</td>
			<td>Bayer&#160;</td>
			<td>PZN 01578681</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">0.9% benzyl alcohol 0.9% saline solution</td>
			<td style="width:215px;">Pharmacy 
			of the University Medical Center Mainz</td>
			<td></td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">gauze (ES-Kompressen)</td>
			<td>Hartmann</td>
			<td>407835</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sterile 5-0 Perma-Hand Silk Suture</td>
			<td>Ethicon Johnson &#38; Johnson</td>
			<td>K890H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ring forceps 1/ 1.5 mm</td>
			<td>Fine Science Tools</td>
			<td>11101-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ring forceps 4.8/ 6 mm</td>
			<td>Fine Science Tools</td>
			<td>11106-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ring forceps 2.2/ 3 mm</td>
			<td>Fine Science Tools</td>
			<td>11103-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adson Forceps-Serrated Straight 12 cm</td>
			<td>Fine Science Tools</td>
			<td>1106-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IrisScissors-Delicate Straight-Sharp/Blunt 10 cm</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mayo-Stille Scissors-Straight 15 cm</td>
			<td>Fine Science Tools</td>
			<td>14012-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Forceps-Inox</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elektroporator CUY21 SC&#160;</td>
			<td>Nepa Gene Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FST 250 Hot Bead Sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microgrinder EG-44</td>
			<td>Narishige</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P-97 Micropette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum electrodes 650P 0.5 mm</td>
			<td>Nepagene</td>
			<td>CUY650P0.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum electrodes 650P 3 mm</td>
			<td>Nepagene</td>
			<td>CUY650P3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum electrodes 650P 5 mm</td>
			<td>Nepagene</td>
			<td>CUY650P5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum electrodes 650P 10 mm</td>
			<td>Nepagene</td>
			<td>CUY650P10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anesthesia system</td>
			<td>Rothacher-Medical GmbH</td>
			<td>CV-30511-3 Vapor 19.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating plate</td>
			<td>Rothacher-Medical GmbH</td>
			<td>HP-1M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature Controller 220V AC</td>
			<td>Rothacher-Medical GmbH</td>
			<td>TCAT-2LV</td>
			<td></td>
		</tr>
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cortex-, hippocampus-, thalamus-, hypothalamus-, lateral septal nucleus- and striatum-specific in utero electroporation in the c57bl/6 mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

الرقم 2، ويبين أمثلة لتحديدا في الرحم Electroporation للمن المناطق النامية في القشرة خلف الطحال، والقشرة الحركية، والقشرة الحسية الجسدية، والقشرة الكمثرية، وقرن آمون 1-3، والتلفيف المسنن، المخطط، نواة الحاجز الجانبية والمهاد وتحت المهاد. وأظهرت نتائج transfections...

Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-، Hippocampus-، Thalamus-، Hypothalamus-، الجانبي الحاجزي Nucleus- والمخطط محددة<em&gt; في الرحم</em&gt; Electroporation للفي C57BL / 6 الماوس

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

The overall goal of this in vivo cell manipulation is to specifically modify cells of certain regions in the central nervous system of the mouse-like cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus, and striatum. This method can help to answer key questions concerning the development and function of the brain, for instance, through the identification of different signaling cascades and the determination of the level of cell differentiation. The main advantage of this technique is that it allows for fast and efficient manipulation of various cell types in the central nervous system without the time-consuming generation of genetically modified mice.Generally, the demonstration of this method is helpful, because individuals new to this method will often struggle with finding the right electrode position, which is critical for specific electroporation. To begin this procedure, cut open the abdominal cavity of an anesthetized pregnant mouse with a scalpel. Prevent the opened abdominal cavity from drying by moistening it with 0.9%warm methyl alcohol saline solution.Then, carefully extract the uterine horns using a pair of ring forceps. Next, position an E14 embryo so that there are no visible vessels above the puncture site. To have a better visualization, use a 5-millimeter electrode to locate the angle and position of the desired area in the brain for transfection.For the in utero electroporation of cortical area, slowly inject 1.5 to two microliters of the DNA solution in the left lateral ventricle. And ensure that the insertion depth is one to two millimeters, measured from the uterine wall. Then, check for blue-green coloring with a sharp demarcation.For better visualization, use a 5-millimeter electrode to locate the angle and position of the desired location in the brain for transfection. After that, place the center of the three-millimeter platinum electrode paddles just anterior of the ear primordia, and make sure that they are not placed above the vessels or the placenta. Subsequently, apply voltage to begin transfection.For the in utero electroporation of hippocampal formation, inject two microliters of DNA solution in the right lateral ventricle of an E15 embryo. Then, place the negative pole of the three to five millimeter platinum electrode paddles just anterior of the ear primordium, and the positive pole at the middle of the ear primordium. Then, apply voltage to begin transfection.For the in utero electroporation of the lateral septal nucleus and striatum, inject one microliter of DNA solution in the right lateral ventricle of an E12 embryo. Then, place the 5-millimeter electrodes at the level of the ear primordia. Subsequently, apply voltage to begin transfection.For the in utero electroporation of the thalamus and hypothalamus, inject one to 1.5 microliters of DNA solution in the lateral ventricle of an E12 to E13 embryo. Then, wait for two to three minutes for the solution to diffuse into the third ventricle. Next, place the 5 or three-millimeter electrodes just posterior to the ear primordia.After that, apply voltage to begin transfection. This schematic shows the appropriate positions for the positive and negative poles for transfecting cortical areas. And this one, shows the positions of the positive and negative pole for transfecting the hippocampal formations.The positions of the positive and negative pole for transfecting the striatum and the lateral septal nucleus are shown here. And the appropriate positions of the electrodes for transfecting the thalamus and the hypothalamus are shown here. The specificity of transfection depends on the electrode size.Increasing the size of electrode paddle reduces the specificity of the transfection. This is especially obvious in younger embryonic stages, but also visible in late embryonic stages. Once mastered, this technique can be done in 15 minutes if it is performed properly.While attempting this procedure, it's important to handle the embryos and the mothers with great care. After watching this video, you should have a good understanding of how to transfect specific regions in the central nervous system of the Black 6 mouse.

Research

JoVE Journal

Neuroscience

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

ترنسفكأيشن من خلايا الشبكية بواسطة الماوس العقدة في الجسم الحي Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

فأر في الرحم Electroporation : الشواهد ترنسفكأيشن جين Spatiotemporal

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

التصور والتلاعب الجيني من التشعبات والاعمده الفقريه في قشرة الدماغ والحصين ماوس باستخدام<em&gt; في الرحم</em&gt; Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

التلاعب الجيني من الفأرة النامية من خلال المهاد<em&gt; في الرحم</em&gt; Electroporation

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

نهج بسيط للحث على العصب الذاتية التجريبية في الفئران C57BL/6 للتقييمات الفنية ولاثيل

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

حمل جزئ لوكس Cre في قشرة الدماغ الماوس عن طريق انهانسر في الرحم

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

جراحة زرع cochlear وتسجيلات استجابة جذع الدماغ السمعية أثارت كهربائياً في الفئران C57BL/6

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

تسجيل LFP في دماغ الفأر للبحث المعرفي وتحليل المخدرات

Dual Extracellular Recordings in the Mouse Hippocampus and Prefrontal Cortex

تسليط الضوء على المؤلف: دراسة تأثيرات الأدوية على إشارات الدماغ باستخدام بروتوكول تسجيل LFP المزدوج في الفئران

تسجيلات مزدوجة خارج الخلية في الحصين الفأر وقشرة الفص الجبهي

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal ...