Name: in utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons
Uploaded: 2010-10-08T17:00:00
Description: Watch this Scientific Journal Video about In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons at JoVE.com

Yazarlar, yararlı tartışmalar için bu tekniği Joseph LoTurco ve Dennis Selkoe teşekkür etmek istiyorum. Yazarlar, bu araştırma desteği için, Amerikan Sağlık Yardım Vakfı bağışçılar teşekkür ederim.

Utero elektroporasyon protokolün temel Kriegstein laboratuar 1, 2 vallahi başka bir makalede ayrıntılı olarak tarif edilmiştir. Bu teknik ilk Osumi laboratuar 3 açıklanan ve bizim protokol LoTurco laboratuar 4 geliştirilen bir dayanmaktadır. Biz protokol nöronal sürecin sonucudur gen fonksiyonu çalışma utero elektroporasyon geçerli olduğu bir açıklama ile takip en önemli ayrıntıları odaklanarak sıçan embriyoların utero elektroporasyon ...

<ol>
	<li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> J Vis Exp. , (2007).</li><li>Walantus, W., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E16+rat+embryos.">In utero intraventricular injection and electroporation of E16 rat embryos.</a> J Vis Exp. , (2007).</li><li>Takahashi, M., Sato, K., Nomura, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+gene+expressions+by+electroporation+in+the+developing+brain+of+mammalian+embryos.">Manipulating gene expressions by electroporation in the developing brain of mammalian embryos.</a> Differentiation. 70 (4-5), 155-1562 (2002).</li><li>Bai, J., Ramos, R. L., Ackman, J. B., Thomas, A. M., Lee, R. V., LoTurco, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+reveals+doublecortin+is+required+for+radial+migration+in+rat+neocortex.">RNAi reveals doublecortin is required for radial migration in rat neocortex.</a> Nat Neurosci. , 1277-1283 (2003).</li><li>Okada, A., Lansford, R., Weimann, J. M., Fraser, S. E., McConnell, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+in+the+developing+nervous+system+with+retrovirus+expressing+modified+green+fluorescent+protein.">Imaging cells in the developing nervous system with retrovirus expressing modified green fluorescent protein.</a> Exp Neurol. 156 (2), 394-406 (1999).</li><li>Bai, J., Ramos, R. L., Paramasivam, M., Siddiqi, F., Ackman, J. B., LoTurco, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+DCX+and+LIS1+in+migration+through+the+lateral+cortical+stream+of+developing+forebrain.">The role of DCX and LIS1 in migration through the lateral cortical stream of developing forebrain.</a> Dev Neurosci. , 144-156 (2008).</li><li>Molyneaux, B. J., Arlotta, P., Menezes, J. R., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+subtype+specification+in+the+cerebral+cortex.">Neuronal subtype specification in the cerebral cortex.</a> Nat Rev Neurosci. , 427-437 (2007).</li><li>Young-Pearse, T. L., Bai, J., Chang, R., Zheng, J. B., Loturco, J. J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Critical+Function+for+-Amyloid+Precursor+Protein+in+Neuronal+Migration+Revealed+by+In+Utero+RNA+Interference.">A Critical Function for -Amyloid Precursor Protein in Neuronal Migration Revealed by In Utero RNA Interference.</a> J Neurosci. 27, 14459-14469 (2007).</li><li>Young-Pearse, T. L., Chen, A. C., Chang, R., Marquez, C., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+APP+regulates+the+function+of+full-length+APP+in+neurite+outgrowth+through+interaction+with+integrin+beta1.">Secreted APP regulates the function of full-length APP in neurite outgrowth through interaction with integrin beta1.</a> Neural Develop. 3, 15-15 (2008).</li></ol>

Primer nöronal kültürlerin in vitro çalışmada neurite akıbet kantitatif analizler için izin verir . Genetik değişiklikler nöronal sürecin sonucudur nasıl etkilediğini araştırmak için, shRNA ya da misexpression yapıları kimyasal transfeksiyon veya viral transdüksiyon yoluyla birincil nöron içine sokulabilir. Ancak, birincil kortikal hücreler ile, hücre tipleri (farklı katmanları, inhibitör nöronlar, glial hücreler glutamaterjik nöronlar) heterojen bir havuz, bu yöntemleri kullanara...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Cortical Neuron Preparation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissection Media:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10X Hanks' Balanced Salt Solution (HBSS) (Ca+2 /Mg +2 free)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14185-052</td>
<td>&nbsp;</td>
<tr>
<td>10X Hanks' Balanced Salt Solution (HBSS) (with Ca+2 /Mg +2 )</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14065-056</td>
<td>&nbsp;</td>
<tr>
<td>1M HEPES pH 7.4</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15630-080</td>
<td>&nbsp;</td>
<tr>
<td>Dishes and Vials:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>100 x 15 mm Petri Dishes</td>
<td>
</td>
<td>Fisherbrand</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>60 x 15 mm Petri Dishes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>351007</td>
<td>&nbsp;</td>
<tr>
<td>15 mL conical vial</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>62-547-205</td>
<td>&nbsp;</td>
<tr>
<td>50 mL conical vial</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>62-554-205</td>
<td>&nbsp;</td>
<tr>
<td>Dissection Tools:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Scissors </td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>91402-12</td>
<td>&nbsp;</td>
<tr>
<td>Standard Forceps</td>
<td>
</td>
<td>Fine Science Tools</td>
<td>11000-12</td>
<td>&nbsp;</td>
<tr>
<td>Curved Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11273-20</td>
<td>&nbsp;</td>
<tr>
<td>Fine Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11255-20</td>
<td>&nbsp;</td>
<tr>
<td>Vannas spring scissors </td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>&nbsp;</td>
<tr>
<td>Miscellaneous:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200</td>
<td>&nbsp;</td>
<tr>
<td>Reichert Bright-Line Hemacytometer</td>
<td>&nbsp;</td>
<td>Hausser Scientific</td>
<td>1490</td>
<td>&nbsp;</td>
<tr>
<td>Hand-Held Tally Counter</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>Z169021</td>
<td>&nbsp;</td>
<tr>
<td>Plating Medium:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Dulbecco's Modified Eagle Medium (D-MEM)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11960-051</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Growth Medium:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>NEUROBASAL Medium </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Serum-Free Supplement </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMAX -I Supplement</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>35050-061</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin Reagent Solution </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15750-060</td>
<td>&nbsp;</td>
<tr>
<td>Immunostaining:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fixative, Washes, and Blocking Buffer:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P6148</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Donkey Serum</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>017-000-121 </td>
<td>&nbsp;</td>
<tr>
<td>Antibodies:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>beta-III tubulin antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB1637</td>
<td>&nbsp;</td>
<tr>
<td>MAP2 antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>AB15452</td>
<td>&nbsp;</td>
<tr>
<td>Donkey Cy3 anti-mouse</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>715-166-151</td>
<td>&nbsp;</td>
<tr>
<td>Donkey Cy2 anti-chicken</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>703-226-155 </td>
<td>&nbsp;</td>
<tr>
<td>DAPI</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>D3571</td>
<td>&nbsp;</td>
<tr>
<td>Slide Preparation:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CC2 Coated Two-Chamber Slides </td>
<td>&nbsp;</td>
<td>Lab-Tek</td>
<td>154852</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent Mounting Media</td>
<td>&nbsp;</td>
<td>KPL</td>
<td>71-00-16</td>
<td>&nbsp;</td>
<tr>
<td>24 x 60 mm Micro Cover Glasses</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>48393-106 </td>
<td>&nbsp;</td>
<tr>
<td>Clear nail polish</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72180</td>
<td>&nbsp;</td>
<tr>
<td>Electroporation:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ketamine</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>995-2949</td>
<td>&nbsp;</td>
<tr>
<td>Xylazine</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>4015809TV</td>
<td>&nbsp;</td>
<tr>
<td>buprenorphine</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>1118217</td>
<td>&nbsp;</td>
<tr>
<td>Picospritzer III</td>
<td>&nbsp;</td>
<td>Parker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BTX square wave electroporator</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BTXECM830</td>
<td>&nbsp;</td>
<tr>
<td>Tweezertrodes, 7 mm, platinum</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>450488</td>
<td>&nbsp;</td>		</tbody>
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in utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons

Primer nöronal kültürlerin in vitro çalışmada, kantitatif analizler için neurite akıbet sağlar . Genetik değişiklikler nöronal sürecin sonucudur, shRNA veya cDNA yapıları kimyasal transfeksiyon veya viral transdüksiyon yoluyla birincil nöron içine sokulabilir nasıl etkilediğini araştırmak için. Ancak, birincil kortikal hücreler ile, hücre tipleri (farklı katmanları, inhibitör nöronlar, glial hücreler glutamaterjik nöronlar) heterojen bir havuz, bu yöntemleri kullanarak transfekte vardır. Utero elektroporasyon embriyonik kemirgen korteks DNA yapıları tanıtmak için kullanılması hedeflenen hücreleri belirli altkümelerini sağlar: elektroporasyon korteksin erken embriyonik korteks hedefleri derin katmanları, geç embriyonik timepoints az elektroporasyon daha yüzeysel katmanları hedefleri. Ayrıca, dorsal karşı ventral-lateral medial korteks bölgelerinin hedef bireysel embriyolar sonuçları başkanları arasında elektrot diferansiyel yerleştirme. Elektroporasyon ardından, transfekte hücreler, disseke ayrışmış, ve neurite akıbet kantitatif analiz için in vitro kaplı olabilir. Burada, kortikal hücrelerin altkümelerini nöronal sürecin sonucudur kantitatif ölçmek için bir adım-adım yöntemi sağlar. Utero elektroporasyon protokolün temel Kriegstein laboratuar 1, 2, diğer iki vallahi makalelerde detaylı olarak tarif edilmiştir . Biz en önemli ayrıntıları odaklanarak nöronal sürecin sonucudur gen fonksiyonu çalışmaya utero elektroporasyon geçerlidir protokolün bir açıklama ile takip utero elektroporasyon için protokol genel bir bakış, sağlayacaktır.

Watch this Scientific Journal Video about In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons at JoVE.com

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

Utero elektroporasyon nöronal progenitör hücrelerinin in vivo transfecting için değerli bir yöntemdir. Elektrotlar ve elektroporasyon gelişimsel timepoint yerleştirme bağlı kortikal hücrelerin bazı altkümelerini hedeflenebilir. Hedeflenen hücreler daha sonra in vivo veya in vitro genetik değişiminin etkileri analiz edilebilir.

In utero Elektroporasyon Kortikal nöronlar Altküme Gen Fonksiyonu Öğrenmenin İlköğretim Nöronal Kültür takip

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

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Neuroscience

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Embriyonik Fare Hipokampal ve Kortikal nöronlar primer Nucleofection ve Kültür

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Verimli kullanarak birden çok CNS Topraklar&#39;da Gen Teslimat In Utero Elektroporasyon

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Fare Utero Elektroporasyon: Kontrollü Spatiotemporal Gen Transfeksiyon

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

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

İlköğretim Rat Kortikal Nöronlar ve Glia bilaminar Co-kültür

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminerjik Nöron Geliştirme Gen Fonksiyonunun Çalışılması için Chick orta beyin içinde ovo Elektroporasyon yılında

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Topikal Transgenik Rats nesil tarafından<em&gt; Utero</em&gt; Elektroporasyon ve<em&gt; In vivo</em&gt; Biyoparlaklık Eleme

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Nöronal Ateşleme Oranlarının Manipülasyon tarafından Astrositik reseptörleri Plastisite Inducing

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

Hayvanlar Rapid Genotipleme Beyin Nöronlar İlköğretim Kültürler kurulması izledi

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Rat Kortikal Nöronlar Filamentli Aktin (F-aktin) puncta Niceleme

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

Rahimde Elektroporasyon Nöronal alt popülasyonlar heyecanlanma ve Tek hücreli Bağlantı Eğitim Yaklaşımları

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...