eoe sub articles

EoE Sub Articles

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&#39;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.

cre-loxp-mediated-induction-cerebral-cavernous-malformations-mouse

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Cerebrovascular Diseases

29 vistas. Fuente: Choi, J. P., et al. Imágenes de inducción y micro-TC de malformaciones cavernosas cerebrales en modelo de ratón. J. Vis. Exp. (2017) En este video, una cría de ratón modificada genéticamente portadora del gen Ccm2 flanqueada por sitios loxP y Cre recombinasa fusionada con un receptor de estrógeno se utiliza para inducir malformaciones cavernosas cerebrales (MCC). La administración de 4-hidroxitamoxifeno activa Cre en las células endoteliales, lo que conduce a la deleción de Cc...

Video: Inducción mediada por Cre-LoxP de malformaciones cavernosas cerebrales en un modelo de ratón - Concepto

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

Un método basado en el Micro-CT para caracterizar las lesiones y la localización de electrodos en cerebros de animales pequeños

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

Investigación

JoVE Journal

Neurociencias

Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System

Brown adipose tissue (BAT) is an adipose depot specialized in energy dissipation that can also serve as an endocrine organ via the secretion of bioactive molecules. The creation of BAT-specific knockout mice is one of the most popular approaches for understanding the contribution of a gene of interest to BAT-mediated energy regulation. The conventional gene targeting strategy utilizing the Cre-LoxP system has been the principal approach to generate tissue-specific knockout mice. However, this approach is time-consuming and tedious. Here, we describe a protocol for the rapid and efficient knockout of a gene of interest in BAT using a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The interscapular BAT is located in the deep layer between the muscles. Thus, the BAT must be exposed in order to inject the AAV precisely and directly into the BAT within the visual field. Appropriate surgical handling is crucial to prevent damage to the sympathetic nerves and vessels, such as the Sultzer's vein that connects to the BAT. To minimize tissue damage, there is a critical need to understand the three-dimensional anatomical location of the BAT and the surgical skills required in the technical steps. This protocol highlights the key technical procedures, including the design of sgRNAs targeting the gene of interest, the preparation of AAV-sgRNA particles, and the surgery for the direct microinjection of AAV into both BAT lobes for generating BAT-specific knockout mice, which can be broadly applied to study the biological functions of genes in BAT.

In this protocol, we describe the technical procedures to generate brown adipose tissue (BAT)-specific knockout mice leveraging a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The described steps include the design of the sgRNAs, the preparation of the AAV-sgRNA particles, and the microinjection of AAV into the BAT lobes.

Generación de ratones knockout específicos de grasa marrón utilizando un sistema combinado de ARN de guía única Cre-LoxP, CRISPR-Cas9 y virus adenoasociado

Brown adipose tissue (BAT) is an adipose depot specialized in energy dissipation that can also serve as an endocrine organ via the secretion of bioactive ...

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

Transcripción

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