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

Autor: Ilchuk LA; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia., Stavskaya NI; Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Street, 119334 Moscow, Russia., Varlamova EA; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia.; 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., Khamidullina AI; Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Street, 119334 Moscow, Russia., Tatarskiy VV; Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Street, 119334 Moscow, Russia., Mogila VA; Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Street, 119334 Moscow, Russia., Kolbutova KB; Head Center of Hygiene and Epidemiology of Federal Medical Biological Agency of Russia, 1st Pekhotniy Pereulok, 6, 123182 Moscow, Russia., Bogdan SA; Head Center of Hygiene and Epidemiology of Federal Medical Biological Agency of Russia, 1st Pekhotniy Pereulok, 6, 123182 Moscow, Russia., Sheremetov AM; Head Center of Hygiene and Epidemiology of Federal Medical Biological Agency of Russia, 1st Pekhotniy Pereulok, 6, 123182 Moscow, Russia., Baulin AN; Head Center of Hygiene and Epidemiology of Federal Medical Biological Agency of Russia, 1st Pekhotniy Pereulok, 6, 123182 Moscow, Russia., Filatova IA; Head Center of Hygiene and Epidemiology of Federal Medical Biological Agency of Russia, 1st Pekhotniy Pereulok, 6, 123182 Moscow, Russia., Silaeva YY; Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov Street, 119334 Moscow, Russia., Filatov MA; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia., Bruter AV; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia.; 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.
Jazyk: angličtina
Zdroj: International journal of molecular sciences [Int J Mol Sci] 2022 Nov 15; Vol. 23 (22). Date of Electronic Publication: 2022 Nov 15.
DOI: 10.3390/ijms232214077
Abstrakt: 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.
Databáze: MEDLINE
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