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

लेखकों के लिए इस तकनीक पर उपयोगी विचार - विमर्श के लिए यूसुफ LoTurco और डेनिस Selkoe धन्यवाद देना चाहूंगा. लेखकों अमेरिकी स्वास्थ्य सहायता फाउंडेशन के दानदाताओं इस शोध के समर्थन के लिए धन्यवाद.

utero electroporation में के लिए बुनियादी प्रोटोकॉल Kriegstein प्रयोगशाला 1, 2 से दूसरे जौव लेख में विस्तार में वर्णित किया गया है. इस तकनीक को मूल रूप से Osumi प्रयोगशाला 3 में वर्णित किया गया था और हमारे प्रोटो...

<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>

प्राथमिक neuronal संस्कृतियों के इन विट्रो अध्ययन में neurite परिणाम की मात्रात्मक विश्लेषण के लिए अनुमति देते हैं . आदेश में अध्ययन करने के लिए कैसे आनुवंशिक परिवर्तन neuronal प्रक्रिया के परिणाम को प्रभावि...

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<td>Cortical Neuron Preparation</td>
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<td>10X Hanks' Balanced Salt Solution (HBSS) (Ca+2 /Mg +2 free)</td>
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<td>10X Hanks' Balanced Salt Solution (HBSS) (with Ca+2 /Mg +2 )</td>
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<td>1M HEPES pH 7.4</td>
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<td>15 mL conical vial</td>
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<td>Scissors </td>
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<td>Fine Science Tools</td>
<td>91402-12</td>
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<td>Vannas spring scissors </td>
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<td>Gibco</td>
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<td>Paraformaldehyde</td>
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<td>Phosphate Buffered Saline</td>
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<td>Donkey Serum</td>
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<td>Jackson Immuno</td>
<td>017-000-121 </td>
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<td>Antibodies:</td>
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<td>beta-III tubulin antibody</td>
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<td>Chemicon</td>
<td>MAB1637</td>
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<td>MAP2 antibody</td>
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<td>Chemicon</td>
<td>AB15452</td>
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<td>Donkey Cy3 anti-mouse</td>
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<td>Donkey Cy2 anti-chicken</td>
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<td>Jackson Immuno</td>
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<td>DAPI</td>
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<td>CC2 Coated Two-Chamber Slides </td>
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<td>71-00-16</td>
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<td>VWR</td>
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<td>Clear nail polish</td>
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<td>&nbsp;</td>
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<td>Xylazine</td>
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<td>Henry Schein</td>
<td>4015809TV</td>
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<td>buprenorphine</td>
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<td>Henry Schein</td>
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<td>Picospritzer III</td>
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<td>Parker</td>
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<td>BTX square wave electroporator</td>
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<td>Fisher</td>
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<td>Tweezertrodes, 7 mm, platinum</td>
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in utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons

प्राथमिक neuronal संस्कृतियों के इन विट्रो अध्ययन में neurite परिणाम की मात्रात्मक विश्लेषण के लिए अनुमति देता है . आदेश में अध्ययन करने के लिए कैसे आनुवंशिक परिवर्तन neuronal प्रक्रिया के परिणाम, shRNA या सीडीएनए constructs रासायनिक अभिकर्मक या वायरल पारगमन के माध्यम से प्राथमिक न्यूरॉन्स में पेश किया जा सकता है है प्रभावित है. हालांकि, प्राथमिक cortical कोशिकाओं के साथ, सेल प्रकार (विभिन्न परतों, निरोधात्मक न्यूरॉन्स, glial कोशिकाओं से glutamatergic न्यूरॉन्स) के एक विषम पूल इन तरीकों का उपयोग ट्रांसफ़ेक्ट हैं. भ्रूण कृंतक प्रांतस्था में डीएनए constructs परिचय utero electroporation में के उपयोग के लिए लक्षित किया जा कोशिकाओं के कुछ सबसेट के लिए अनुमति देता है: जबकि जल्दी प्रांतस्था के भ्रूण प्रांतस्था लक्ष्य गहरी परतों के electroporation देर भ्रूण timepoints पर electroporation अधिक सतही परतों लक्ष्य. इसके अलावा, प्रांतस्था के पृष्ठीय औसत दर्जे बनाम उदर - पार्श्व क्षेत्रों का लक्ष्यीकरण में व्यक्तिगत भ्रूण परिणामों के सिर में इलेक्ट्रोड के अंतर स्थान. Electroporation के बाद, ट्रांसफ़ेक्ट कोशिकाओं बाहर dissected किया जा सकता है, अलग, और neurite परिणाम के मात्रात्मक विश्लेषण के लिए इन विट्रो में मढ़वाया. यहाँ, हम एक कदम दर कदम विधि करने के लिए मात्रात्मक cortical कोशिकाओं के सबसेट में neuronal प्रक्रिया परिणाम उपाय प्रदान करते हैं. utero electroporation में के लिए बुनियादी प्रोटोकॉल Kriegstein प्रयोगशाला 1, 2 से दो अन्य जौव लेख में विस्तार में वर्णित किया गया है. हम utero electroporation में के लिए हमारे प्रोटोकॉल के एक सिंहावलोकन है, सबसे महत्वपूर्ण विवरण पर ध्यान केंद्रित, हमारे प्रोटोकॉल है कि utero electroporation में neuronal प्रक्रिया के परिणाम में जीन समारोह के अध्ययन के लिए लागू होता है की एक विवरण के द्वारा पीछा प्रदान करेगा.

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 electroporation vivo में neuronal पूर्वपुस्र्ष कोशिकाओं transfecting के लिए एक मूल्यवान तरीका है . इलेक्ट्रोड और electroporation की विकास timepoint के स्थान पर निर्भर करता है, cortical कोशिकाओं के कुछ सबसेट को लक्षित किया जा सकता है. लक्षित कोशिकाओं तो vivo में या आनुवंशिक परिवर्तन के प्रभाव के लिए इन विट्रो में विश्लेषण किया जा सकता है.

Utero में electroporation cortical न्यूरॉन्स के सबसेट में जीन समारोह का अध्ययन करने के लिए प्राथमिक Neuronal संस्कृति द्वारा पीछा

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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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

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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

एकाधिक सीएनएस का प्रयोग शासित प्रदेशों में कुशल जीन डिलीवरी Utero में Electroporation

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

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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

प्राथमिक चूहा cortical न्यूरॉन्स और Glia Bilaminar सह संस्कृति

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

चिकी midbrain डोपामिनर्जिक न्यूरॉन विकास में जीन समारोह के अध्ययन के लिए ओवो electroporation में

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

Topically ट्रांसजेनिक चूहों की पीढ़ी द्वारा<em&gt; Utero में</em&gt; Electroporation और<em&gt; में विवो</em&gt; Bioluminescence स्क्रीनिंग

 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

Neuronal फायरिंग दरों के हेरफेर से astrocytic रिसेप्टर्स की Plasticity उत्प्रेरण

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

पशु की रैपिड जीनोटाइपिंग मस्तिष्क न्यूरॉन्स के प्राथमिक संस्कृतियों की स्थापना के द्वारा पीछा किया

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

चूहा cortical न्यूरॉन्स में filamentous actin (एफ actin) puncta की मात्रा

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

गर्भाशय में Electroporation Neuronal उप-जनसंख्या के excitability और एकल कोशिका कनेक्टिविटी अध्ययन के लिए दृष्टिकोण

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