Name: transfecting human neural stem cells with the amaxa nucleofector
Uploaded: 2007-07-19T00:00:00
Description: Watch this Scientific Journal Video about Transfecting Human Neural Stem Cells with the Amaxa Nucleofector at JoVE.com

<p class="jove_content">The authors would like to acknowledge Dr. Philip H. Schwartz of the National Human Neural Stem Cell Resource at the Children s Hospital, Orange County Research Institute for providing hNSPC cultures.</p>

<p class="jove_content"><strong>Note: </strong>Refer to the <strong>Passaging Human Neural Stem Cells</strong> article (<a href="https://www.jove.com/index/Details.stp?ID=263">https://www.jove.com/index/Details.stp?ID=263</a>) and the <strong>Counting Human Neural Stem Cells</strong> article (<a href="https://www.jove.com/index/Details.stp?ID=262">https://www.jove.com/index/Details.stp?ID=262</a>) to learn how to resuspend and count hNSPCs.</p>
<p class="jove_step">Preparation</p>
<ol>
<li>Make sure that there are plenty of cells available for ...

<ol>
	<li>Gresch, O., Engel, F. B., Nesic, D., Tran, T. T., England, H. M., Hickman, E. S., Krner, I., Gan, L., Chen, S., Castro-Obregon, S., Hammermann, R., Wolf, J., M ller-Hartmann, H., Nix, M., Siebenkotten, G., Kraus, G., Lun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+non-viral+method+for+gene+transfer+into+primary+cells.">New non-viral method for gene transfer into primary cells.</a> <em style='font-style: italic'>Methods</em>. <b>33</b>, 151-163 (2004).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+Human+Neural+Stem+Cells.">Passaging Human Neural Stem Cells.</a> <em style='font-style: italic'>Journal of Visualized Experiments</em>. <b>7</b>, (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+Human+Neural+Stem+Cells.">Counting Human Neural Stem Cells.</a> <em style='font-style: italic'>Journal of Visualized Experiments</em>. <b>7</b>, (2007).</li></ol>

<p class="jove_content">This protocol describes a relatively rapid and efficient transfection procedure for hNSPCs.  Using this procedure, we have obtained initial transfection efficiencies of ~35%.  Cells transfected by this procedure can be used for short-term studies (transiently transfected cells) or for longer-term studies if a selection agent is used to generate a stably transfected population.  We have generated stably transfected cells in which ~70% of the cells express the exogenous protein.  In transiently transfected hNSPCs, we have ...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>The Nucleofector Device</td>
<td>Tool</td>
<td>Amaxa</td>
<td>AAD-1001</td>
<td>&nbsp;</td>
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<td>Mouse neural stem cell nucleofector kit</td>
<td>Reagent</td>
<td>Amaxa</td>
<td>VPG-1004</td>
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transfecting human neural stem cells with the amaxa nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Watch this Scientific Journal Video about Transfecting Human Neural Stem Cells with the Amaxa Nucleofector at JoVE.com

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

<p class="jove_content">Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo.  This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.</p>

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Research

JoVE Journal

Biology

Counting Human Neural Stem Cells

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Passaging Human Neural Stem Cells

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