Name: isolation and derivation of mouse embryonic germinal cells
Uploaded: 2009-10-22T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Derivation of Mouse Embryonic Germinal Cells at JoVE.com

<p class="jove_content">The authors would like to acknowledge the invaluable help of:
Dr Neal First and Kourtney Wilkinson for manuscript editing; Dr Lucy Senter, Dr. Brigit Willeford and Mike Basett for assistance with training and animal care at the MSU ALAC accredited mouse facility; Dr Dwayne Wise for his assistance with microscopy and image capture; Cesar Monroy, Hannah Swoope, and Bobbie Huddleston for their assistance with video production at MSU. This research was funded by Office of Institutional Research and the Department of Biological Sciences at Mississippi State University.</p>

<p class="jove_title">Part 1: Pregnant Mouse Laparotomy</p>
<ol>
  <li>Using cervical dislocation, euthanize a pregnant C57BL6J female mouse at 10.5-11.5 dpc.</li>
  <li>Clean the abdomen with antimicrobial soap, and then, shave it.</li>
  <li>After shaving, wash the abdomen with a saline solution. </li>
  <li>Then, dry the abdomen with a sterile gauze.</li>
  <li>Cover the mouse with a new sterile field.</li>
  <li>Make a ventral incision using forceps and dissection scissors.</li>
  <li>Identify and remove the entire uterus from the a...

<ol>
	<li>DeFelici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Cell Biology: A laboratory Handbook</em>. <b>1</b>, 73-85 (1998).</li><li>Donovan, P. J., De Miguel, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+Germ+Cells+into+Stem+Cells.">Turning Germ Cells into Stem Cells.</a> <em style='font-style: italic'>Current Opinion in Genetics and Development</em>. <b>13</b>, 463-471 (2003).</li><li>Labosky, P. A., Hogan, B. L. M., Sharp, P. T., Mason, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Part+12:+Mouse+Primordial+Germ+Cells:+isolation+and+in+vitro+culture.">Part 12: Mouse Primordial Germ Cells: isolation and in vitro culture.</a> <em style='font-style: italic'>Molecular embryology: methods and protocols</em>. <b>97</b>, (1999).</li><li>Mc Laren, A., Southee, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+Mouse+Embryonic+Germ+Cells+into+Meiosis.">Entry of Mouse Embryonic Germ Cells into Meiosis.</a> <em style='font-style: italic'>Developmental Biology</em>. <b>187</b>, 107-113 (1997).</li><li>Yoshimizu, T., Obinata, M., Matsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+tissue+and+cell+interactions+play+key+roles+in+mouse+germ+cell+specification.">Stage-specific tissue and cell interactions play key roles in mouse germ cell specification.</a> <em style='font-style: italic'>Development</em>. <b>128</b>, 481-490 (2001).</li><li>Zhang, J., Hhvorostov, I., Teitell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+MEFs+to+Matrigel+I:+Passaging+hESCs+in+the+Presence+of+MEFs.">From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs.</a> <em style='font-style: italic'>J Vis Exp</em>. , (2008).</li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>10.5 - 11.5 dpc pregnant C57BL6J female mouse crossed with the same background male mouse.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mouse Fibroblast MEF-CF1 ATCC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>SCRC-1040</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Phosphate Buffered Saline (D-PBS)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190/086</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase /Dispase Solution</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>269638</td>
<td>&nbsp;</td>
<tr>
<td>Gelatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1890</td>
<td>&nbsp;</td>
<tr>
<td>Mitomycin C</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M4287</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-072</td>
<td>&nbsp;</td>
<tr>
<td>MEF Medium: Dulbecco Medium Eagle Modified (D-MEM) GLUTAMAX High glucose</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10566024</td>
<td>&nbsp;</td>
<tr>
<td>10% Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000/044</td>
<td>&nbsp;</td>
<tr>
<td>1% of antibiotic-antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15420/096</td>
<td>&nbsp;</td>
<tr>
<td>Knockout Media: 80% knockout D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10829/018</td>
<td>&nbsp;</td>
<tr>
<td>20% Knockout serum Invitrogen Cat. No. 1028/028, 200mM L-glutamine Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>25130/081</td>
<td>with &beta;-mercaptoethanol Sigma Catalogue Number: M7522</td>
</tr>
<tr>
<td>1X non essential amino acid solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140/050</td>
<td>&nbsp;</td>
<tr>
<td>1% antibiotic-antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15420/096</td>
<td>supplemented with grow factor as 2500 U of Leukemia inhibitory factor (mLIF-ESGRO) Millipore Catalogue Number: ESG1106</td>
</tr>
<tr>
<td>40 ng/ml of Recombinant Murine Stem Cell Factor (SCF) ReproTech</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>250-03</td>
<td>&nbsp;</td>
<tr>
<td>20 ng/ml of Basic Fibroblast Growth factor (bFGF)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13256-029</td>
<td>&nbsp;</td>
<tr>
<td>Corning center well culture dish 60mm</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>07-200-79</td>
<td>1.5 ml eppendorf tube, and 15 ml falcon tubes</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
			Forceps No. 14 and 15, scalpel 35 mm, dissection scissors, surgical fields, antimicrobial soap, saline solution 0.9 %, gauze, ice foam container, razor blade, scalpel 35 mm, fine forceps No. 4 and 5, fine teasing needle with handle, filter paper sheets, and pulled glass pipettes.<br /><br />Equipment: dissecting stereomicroscope, light source Schott Fostec, inverted microscope, micropipette 10 and 200ul, centrifuge, bio safety Cabinet.		</div>
		

isolation and derivation of mouse embryonic germinal cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

Watch this Scientific Journal Video about Isolation and Derivation of Mouse Embryonic Germinal Cells at JoVE.com

Isolation and Derivation of Mouse Embryonic Germinal Cells

<p class="jove_content">The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.</p>

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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