<p class='jove_content'>All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.</p>
<p class='jove_title'><strong>1. Induction of Transfected CreER with Tamoxifen</strong></p>
<p class='jove_content'><strong>CAUTION:</strong> Tamoxifen is harmful, may be cancerous or damage fertility. Please refer to the safety data sheet.</p>
<ol>
	<li>Plan intraperitoneal tamoxifen injections on three consecutive days.</li>
	<li>On day 1, dissolve 10 mg of tamoxifen in 40 &#181;L ethanol. Incubate at 55 &#176;C for 10 min. Vortex several times until the tamoxifen has dissolved.</li>
	<li>Add 960 &#181;L corn oil. Incubate for 5 mins at 55 &#176;C. Vortex several times to get a clear solution.</li>
	<li>Prepare the solution in a 1-mL insulin syringe with a 27 G needle.</li>
	<li>Scruff the mouse by grabbing the neck of the mouse carefully with the thumb and the second finger, fixing the tail between the base of the hand and the fourth and fifth finger.</li>
	<li>Inject 0.1 mL (= 1 mg of tamoxifen) of the solution intraperitoneally into the left lower quadrant of the abdomen.</li>
	<li>Repeat the injections on days two and three.</li>
</ol>

<table><tbody><tr><td>Injekt Syringe F 1 ml</td><td>&amp;nbsp;Braun&amp;nbsp;</td><td>9166017V&amp;nbsp;</td><td>For intraperitoneal injection</td></tr><tr><td>Tamoxifen&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T5648-1G&amp;nbsp;</td><td>For CreER activation</td></tr><tr><td>Corn oil&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C8267-500ML&amp;nbsp;</td><td>Carrier for tamoxifen injections</td></tr></tbody></table>

creer-loxp system-based target gene inactivation: a tamoxifen-inducible cre recombinase system for target gene knockout in a mouse model

CreER-LoxP System-Based Target Gene Inactivation: A Tamoxifen-Inducible Cre Recombinase System for Target Gene Knockout in a Mouse Model

<p class='jove_content'>Source: Hubner, E. K., <em>et al.</em> <a href='https://www.jove.com/v/56613'>Constitutive and Inducible Systems for Genetic&#160;<em>In Vivo&#160;</em>Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection.</a>&#160;<em>J. Vis. Exp. </em>(2018)</p>
<p class='jove_content'>This video describes a procedure for target gene knockout using a tamoxifen-inducible Cre-recombinase system following intraperitoneal injection of tamoxifen in a mouse model.</p>

Source: Hubner, E. K., et al. Constitutive and Inducible Systems for Genetic&#160;In Vivo&#160;Modification of Mouse Hepatocytes Using Hydrodynamic Tail ...

<p>Begin with a genetically engineered mouse model comprising a modified genome that harbors LoxP sites - specific sequences recognized by Cre-recombinase enzymes - flanking the gene of interest. The mouse is further transfected with a plasmid vector encoding organ-specific CreER recombinase, leading to the integration of CreER recombinase-expressing element into the genome of liver cells.</p>
<p>The expressed CreER recombinase, an inactive fusion protein involving Cre recombinase and the mutant ligand-binding domain of the estrogen receptor, ER, interacts and binds to heat-shock protein in the cytoplasm. This binding confines CreER recombinase in the cytoplasm.</p>
<p>Prepare a solution of tamoxifen, a hydrophobic drug, using ethanol and suitable oil. Now, inject the tamoxifen solution intraperitoneally into the left lower quadrant of the mouse's abdomen to prevent damage to the abdominal organs.</p>
<p>The injected tamoxifen is metabolized in the liver to 4-hydroxytamoxifen, 4-OHT. Further, 4-OHT, a selective estrogen receptor modulator, binds to the ER-ligand binding domain of CreER recombinase, causing its conformational change. This leads to the release of the CreER recombinase from the heat-shock protein.</p>
<p>The activated CreER recombinases translocate into the nucleus and recognize the LoxP sites in the host genome. The CreER recombinases catalyze site-specific recombination between the LoxP sites, resulting in the excision of the gene of interest, thereby achieving tissue-specific gene knockout.</p>

<p>Initiate induction of Cre Recombinase 10 to 15 days after tail vein injection, to allow for clearance of non-integrated vectors. Dissolve 10 milligrams of tamoxifen in 40 microliters of ethanol, and incubate at 55 degrees Celsius for 10 minutes with shaking.</p>
<p>Vortex several times until the tamoxifen has dissolved, then add 960 microliters of corn oil, and incubate for 5 minutes at 55 degrees Celsius. After cooling, draw the solution into a 1-milliliter insulin syringe with a 20-gauge needle, then change to a 27-gauge needle.</p>
<p>To inject, first, scruff the mouse by grabbing the neck of the mouse carefully with the thumb and the second finger, fixing the tail between the base of the hand and the fourth and fifth finger, then, intraperitoneally inject 0.1 milliliters of the solution into the left lower quadrant of the abdomen.</p>

creer-loxp-system-based-target-gene-inactivation-tamoxifen-inducible

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Gene knock-out

7.1K Views. Begin with a genetically engineered mouse model comprising a modified genome that harbors LoxP sites - specific sequences recognized by Cre-recombinase enzymes - flanking the gene of interest. The mouse is further transfected with a plasmid vector encoding organ-specific CreER recombinase, leading to the integration of CreER recombinase-expressing element into the genome of liver cells. The expressed CreER recombinase, an inactive fusion protein involving Cre recombinase and the mutant...

Abstract

Protocol

Disclosures

<table><tbody><tr><td>Injekt Syringe F 1 ml</td><td>&nbsp;Braun&nbsp;</td><td>9166017V&nbsp;</td><td>For intraperitoneal injection</td></tr><tr><td>Tamoxifen&nbsp;</td><td>Sigma-Aldrich</td><td>T5648-1G&nbsp;</td><td>For CreER activation</td></tr><tr><td>Corn oil&nbsp;</td><td>Sigma-Aldrich</td><td>C8267-500ML&nbsp;</td><td>Carrier for tamoxifen injections</td></tr></tbody></table>

CreER-LoxP System-Based Target Gene Inactivation: A Tamoxifen-Inducible Cre Recombinase System for Target Gene Knockout in a Mouse Model

In This Article

Abstract

Protocol

Disclosures

Materials

Tags

Name	Company	Catalog Number	Comments
Injekt Syringe F 1 ml	Braun	9166017V	For intraperitoneal injection
Tamoxifen	Sigma-Aldrich	T5648-1G	For CreER activation
Corn oil	Sigma-Aldrich	C8267-500ML	Carrier for tamoxifen injections