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Article

Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System

by
Leonid A. Ilchuk
1,†,
Nina I. Stavskaya
2,†,
Ekaterina A. Varlamova
1,3,
Alvina I. Khamidullina
2,
Victor V. Tatarskiy
2,
Vladislav A. Mogila
2,
Ksenia B. Kolbutova
4,
Sergey A. Bogdan
4,
Alexey M. Sheremetov
4,
Alexandr N. Baulin
4,
Irina A. Filatova
4,
Yulia Yu. Silaeva
2,
Maxim A. Filatov
1,* and
Alexandra V. Bruter
1,3
1
Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia
2
Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Street, 119334 Moscow, Russia
3
Federal State Budgetary Institution “N.N. Blokhin National Medical Research Center of Oncology”, Ministry of Health of the Russian Federation, Kashirskoe Sh., 24, 115478 Moscow, Russia
4
Head Center of Hygiene and Epidemiology of Federal Medical Biological Agency of Russia, 1st Pekhotniy Pereulok, 6, 123182 Moscow, Russia
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2022, 23(22), 14077; https://doi.org/10.3390/ijms232214077
Submission received: 16 October 2022 / Revised: 10 November 2022 / Accepted: 11 November 2022 / Published: 15 November 2022
(This article belongs to the Special Issue Transgenic Mice in Human Diseases: Insights from Molecular Research 3.0)
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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/ERT2 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 Cdk8floxed/floxed/Rosa-Cre-ERT2 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.
Keywords: Cre-LoxP; Cre-ERT2; tamoxifen; genetically engineered mice; genome-edited mice; conditional knockout; cyclin-dependent kinases 8/19 Cre-LoxP; Cre-ERT2; tamoxifen; genetically engineered mice; genome-edited mice; conditional knockout; cyclin-dependent kinases 8/19

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MDPI and ACS Style

Ilchuk, L.A.; Stavskaya, N.I.; Varlamova, E.A.; Khamidullina, A.I.; Tatarskiy, V.V.; Mogila, V.A.; Kolbutova, K.B.; Bogdan, S.A.; Sheremetov, A.M.; Baulin, A.N.; et al. Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System. Int. J. Mol. Sci. 2022, 23, 14077. https://doi.org/10.3390/ijms232214077

AMA Style

Ilchuk LA, Stavskaya NI, Varlamova EA, Khamidullina AI, Tatarskiy VV, Mogila VA, Kolbutova KB, Bogdan SA, Sheremetov AM, Baulin AN, et al. Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System. International Journal of Molecular Sciences. 2022; 23(22):14077. https://doi.org/10.3390/ijms232214077

Chicago/Turabian Style

Ilchuk, Leonid A., Nina I. Stavskaya, Ekaterina A. Varlamova, Alvina I. Khamidullina, Victor V. Tatarskiy, Vladislav A. Mogila, Ksenia B. Kolbutova, Sergey A. Bogdan, Alexey M. Sheremetov, Alexandr N. Baulin, and et al. 2022. "Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System" International Journal of Molecular Sciences 23, no. 22: 14077. https://doi.org/10.3390/ijms232214077

APA Style

Ilchuk, L. A., Stavskaya, N. I., Varlamova, E. A., Khamidullina, A. I., Tatarskiy, V. V., Mogila, V. A., Kolbutova, K. B., Bogdan, S. A., Sheremetov, A. M., Baulin, A. N., Filatova, I. A., Silaeva, Y. Y., Filatov, M. A., & Bruter, A. V. (2022). Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System. International Journal of Molecular Sciences, 23(22), 14077. https://doi.org/10.3390/ijms232214077

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