Name: nuclear transfer into mouse oocytes
Uploaded: 2006-11-30T00:00:00
Description: Watch this Scientific Journal Video about Nuclear Transfer into Mouse Oocytes at JoVE.com

<p class="jove_content">DE would like to thank Dr. Hide Akutsu for sharing his NT tricks and Dr. Stephen Sullivan and Garrett Birkhoff for critical reading of the protocol.</p>

<p class="jove_title">Preparations:</p>
<ol>
<li class="jove_step">A detailed description of superovulation and microdrop embryo culture can be found elsewhere <sup>2</sup>. </li>
<li class="jove_step">Prepare a petri dish with microdrops of embryo culture medium (e.g. KSOM, Chemicon). Cover with mineral oil (mineral oil batches should be tested for compatibility with embryo culture). Equilibrate at 37&deg;C in air plus 5% CO<sub>2</sub>. (In Thermo-Electron 3110 water jacketed incubator or equivalent)</li>
<li class="jove_step">Prepare a dish ...

<ol>
	<li>Eggan, K., Jaenisch, R., Pease, S., Lois, C. <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'>Mammalian and Avian Transgenesis- New Approaches</em>. , (2006).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R., Press, C. S. H. L. <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'>Manipulating the mouse embryo</em>. , (2003).</li><li>Wakayama, T., Perry, A. C., Zuccotti, M., Johnson, K. R., Yanagimachi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full-term+development+of+mice+from+enucleated+oocytes+injected+with+cumulus+cell+nuclei.">Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.</a> <em style='font-style: italic'>Nature</em>. <b>394</b>, 369-374 (1998).</li></ol>

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		<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>Cytochalastin B</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>100x stock = 500 ug/mL Cytochalasin B in DMSO.  Store at -20&#x02DA; C.</td>
</tr>
<tr>
<td>Strontium Chloride </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10x stock = 100 mM SrCl2 in H2O.  Store at room temperature.
</td>
</tr>
<tr>
<td>MCZB Stock Salts</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Filter sterilize 500 mL master salts  (good for 3-4 months; store at 4&#x02DA; C)
</td>
</tr>
<tr>
<td>HCZB Stock Salts</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Start with 500 mL master salts. Add 50 mg PVA. Stir for 30-60 min and sterile filter (good for 3 months; store at 4&#x02DA; C).


</td>
</tr>
<tr>
<td>PVA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-8136</td>
<td>cold-soluble</td>
</tr>
<tr>
<td>HCZB with 11% w/v PVP</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Start with 20 ml HCZB medium in a 50 mL conical tube. Place 22g PVP on top of liquid. Close tube and store undisturbed at 4&#x02DA; C for 72 &#x2013; 96 hrs, at which time the PVP will have entered solution. Filter sterilize through an 8 mm filter and store at 4&#x02DA; C.</td>
</tr>
<tr>
<td>PVP</td>
<td>&nbsp;</td>
<td>ICN Biochemicals</td>
<td>&nbsp;</td>
<td>MW 360000</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
			Please check for additional buffers compositions in the Protocol part.

		</div>
		

nuclear transfer into mouse oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

Watch this Scientific Journal Video about Nuclear Transfer into Mouse Oocytes at JoVE.com

Nuclear Transfer into Mouse Oocytes

<p class="jove_content">This movie and the protocol are intended to help learning nuclear transfer.</p>

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

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