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Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ER^T2 System

Leonid A Ilchuk¹, Nina I Stavskaya², Ekaterina A Varlamova^1,3

¹Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia.

International Journal of Molecular Sciences

|November 26, 2022

Abstract:

Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ER^T2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen-the active metabolites of tamoxifen-measured by LC-MS in these tissues. We also describe two cases of Cdk8^{floxed/floxed}/Rosa-Cre-ER^T2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models.

Insights

Tamoxifen-inducible Cre systems show variable knockout efficiency due to metabolite levels. Doxycycline-induced Cre systems are proposed as a more reliable alternative for conditional gene manipulation in mice.

Area of Science:

Genetics
Molecular Biology
Developmental Biology

Background:

Inducible Cre-dependent systems are crucial for conditional gene manipulation in mice.
Doxycycline-dependent transcription and tamoxifen-dependent Cre/ER^T2 translocation are common induction strategies.
Both methods have limitations affecting gene knockout efficiency and applicability.

Purpose of the Study:

To analyze the efficiency of tamoxifen-induced Cre-lox recombination in different mouse tissues.
To investigate the correlation between tamoxifen metabolite concentrations and knockout efficiency.
To identify limitations of tamoxifen-inducible systems and propose alternatives.

Main Methods:

Liquid chromatography-mass spectrometry (LC-MS) for quantifying tamoxifen metabolites (hydroxytamoxifen and endoxifen).
Analysis of Cre-lox recombination efficiency in various tissues of Cdk8^{floxed/floxed}/Rosa-Cre-ER^T2 mice.
Comparative assessment of standard and modified tamoxifen administration protocols.

Main Results:

Tamoxifen-induced knockout efficiency correlates with tissue-specific concentrations of active tamoxifen metabolites.
Standard tamoxifen administration resulted in incomplete knockout in the brain and uterus.
Increased tamoxifen dosage and duration improved brain knockout but not uterine knockout.
Tamoxifen administration during embryonic development negatively impacted gestation, hindering knockout induction.

Conclusions:

Tamoxifen metabolite levels significantly influence Cre-lox recombination efficiency in a tissue-dependent manner.
Tamoxifen-inducible systems present limitations for complete gene knockout in specific tissues and during embryonic development.
Doxycycline-inducible Cre systems are recommended as a more reliable alternative for certain conditional gene editing models.

Read full analysis

Keywords:

Cre-ERT2 Cre-LoxP conditional knockout cyclin-dependent kinases 8/19 genetically engineered mice genome-edited mice tamoxifen

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