All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160;&#160;&#160;
1. Induction of Cerebral Cavernous Malformations or CCMs in Mouse Models
<ol>
	<li>Cross Cdh5-CreErt2; Ccm2fl/fl mice with Ccm2fl/fl to generate litters with Cdh5-CreErt2; Ccm2fl/fl (Ccm2iECKO) pups and littermate controls (Ccm2fl/fl). Cdh5-CreErt2 and Ccm2fl/fl animals have been previously described19.</li>
	<li>Dissolve 4HT in 100% ethanol and store at -80 &#176;C in aliquots of 30 &#181;L 4-hydroxytamoxifen(4HT)( concentration: 10 mg/mL). On the day of use, dilute aliquoted 4HT in corn oil (0.5 mg/mL).</li>
	<li>Intragastrically inject 50 &#181;L of 4HT (0.5 mg/mL) to neonatal pups at P1 to induce experimental CCM lesions using an insulin syringe.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-hydroxy tamoxifen</td>
			<td>Sigma-Aldrich</td>
			<td>H6278</td>
			<td>To activate Cdh5-CreErt2</td>
		</tr>
		<tr>
			<td>Corn oil</td>
			<td>Sigma-Aldrich</td>
			<td>C8267-500ML</td>
			<td>To dilute 4-hydroxy tamoxifen</td>
		</tr>
		<tr>
			<td>Insulin syringe&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

cre-loxp mediated induction of cerebral cavernous malformations in a mouse model

Source: Choi, J. P., et al. <a href='https://app.jove.com/v/56476'>Induction and micro-CT imaging of cerebral cavernous malformations in mouse model.</a> J. Vis. Exp. (2017)
In this video, a genetically modified mouse pup carrying the Ccm2 gene flanked by loxP sites and Cre recombinase fused to an estrogen receptor is used to induce cerebral cavernous malformations (CCMs). The administration of 4-hydroxytamoxifen activates Cre in endothelial cells, leading to the deletion of Ccm2 and the formation of CCMs in the brain.

Cre-LoxP Mediated Induction of Cerebral Cavernous Malformations in a Mouse Model

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review ...

Begin with a genetically modified mouse pup carrying the CCM2 gene, which is essential for blood vessel stability.
This gene is flanked by two loxP sites. The pup's endothelial cells also express Cre recombinase enzyme fused to an estrogen receptor or ER.
Intragastrically inject 4-hydroxytamoxifen or 4-HT into the pup.
The 4-HT diffuses through endothelial cells and binds to the estrogen receptor portion of the Cre-ER fusion protein.
This binding triggers a conformational change that activates Cre recombinase, which enters the nucleus of endothelial cells and binds to the loxP sites.
This facilitates the recombination event between the loxP sites, forming a loop and excises the CCM2 gene segment.
The loss of CCM2 disrupts the integrity of endothelial cells,&#160; compromising the stability of blood vessel walls.
This leads to the development of swollen abnormal vascular structures known as cerebral cavernous malformations or CCMs, which are fragile and prone to leakage.

Begin by dissolving 4-HT in 100% ethanol to a concentration of 10 milligrams per milliliter. Prepare 30 microliter aliquots, and then store at minus 80 Celsius. On the day of use, dilute the aliquoted 4-HT in corn oil to 0.5 milligrams per milliliter. Then, use an insulin syringe to intragastrically inject 50 microliters of 4-HT into P1 neonatal pups to induce experimental CCM lesions.

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756 Views. Begin with a genetically modified mouse pup carrying the CCM2 gene, which is essential for blood vessel stability. This gene is flanked by two loxP sites. The pup's endothelial cells also express Cre recombinase enzyme fused to an estrogen receptor or ER. Intragastrically inject 4-hydroxytamoxifen or 4-HT into the pup. The 4-HT diffuses through endothelial cells and binds to the estrogen receptor portion of the Cre-ER fusion protein. This binding triggers ...

Cre-LoxP Mediated Induction of Cerebral Cavernous Malformations in a Mouse Model

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Name	Company	Catalog Number	Comments
4-hydroxy tamoxifen	Sigma-Aldrich	H6278	To activate Cdh5-CreErt2
Corn oil	Sigma-Aldrich	C8267-500ML	To dilute 4-hydroxy tamoxifen
Insulin syringe