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

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 t...

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Bibliographic Details
Published inInternational journal of molecular sciences Vol. 23; no. 22
Main Authors Ilchuk, Leonid A, Stavskaya, Nina I, Varlamova, Ekaterina A, Khamidullina, Alvina I, Tatarskiy, Victor V, Mogila, Vladislav A, Kolbutova, Ksenia B, Bogdan, Sergey A, Sheremetov, Alexey M, Baulin, Alexandr N, Filatova, Irina A, Silaeva, Yulia Yu, Filatov, Maxim A, Bruter, Alexandra V
Format Journal Article
LanguageEnglish
Published Switzerland 15.11.2022
Subjects
Animals
Doxycycline - pharmacology
Female
Gene Editing - methods
Integrases - genetics
Integrases - metabolism
Mice
Mice, Transgenic
Tamoxifen - pharmacology
cyclin-dependent kinases 8/19
genome-edited mice
Cre-ERT2
conditional knockout
Cre-LoxP
tamoxifen
genetically engineered mice
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Summary: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 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 /Rosa-Cre-ER 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.
ISSN:1422-0067