Transgenic mice Cre-dependently expressing mutant polymerase-gamma: novel test-system for pharmacological study of mitoprotective drugs

Авторы

DOI:

https://doi.org/10.3897/rrpharmacology.7.72784

Аннотация

Introduction: PolG-alpha is a nuclear-encoded enzyme which provides replication and repair of mitochondrial DNA. D257A mutation of PolG-alpha leads to change in the N-terminal ”proofreading” domain, which deprives the enzyme of 3′-5′ exonuclease activity, resulting in accumulation of mutations in the mitochondrial genome.

Materials and methods: Murine zygotes were microinjected with transgene construction carrying mutant murine Polg coding sequence and GFP coding sequence by a loxP-flanked STOP-cassette. Two Cre-activator strains, CMV-Cre (systemic activation) and Tie2-Cre (endothelial activation), were used for activation of the transgene. To confirm the insertion and Cre-dependent activation of the transgene, genotyping and qPCR copy number measurement of mutant Polg were performed, and GFP fluorescence was assessed.

Results: Two primary transgenic animals were used as the founders for two lines with copy numbers of transgene ~7 and ~5. After systemic activation, the number of the transgene copies decreases to ~1.0 while endothelial specific activation does not affect the number of transgene copies in tail tissue.

Discussion: A murine model with spatial control of mutant Polgexpression has been developed. To our knowledge, this is the first transgenic model of tissue-specific mitochondrial dysfunction.

Conclusion: Transgenic mice Cre-dependent expressing mutant polymerase-gamma are a novel test-system for studying mitochondrial biology and efficacy of mitoprotective drugs.

Ключевые слова:

Cre-LoxP system, genome editing, IVF, mitochondrial polymerase-gamma, transgene copy number determination, transgenic mice

Вклад авторов

Marina V. Kubekina, Institute of Gene Biology

Postgraduate student, junior researcher at the Institute of Gene Biology, Russian Academy of Sciences, e-mail: kubekina@genebiology.ru, ORCID ID https://orcid.org/0000-0002-8834-1111. Writing the article, molecular cloning, analyzing transgenic mice PCR, and measuring copy number.

Yulia Yu. Silaeva, Institute of Gene Biology

Ph.D, research fellow at the Institute of Gene Biology, Russian Academy of Sciences, e-mail: yulya.silaeva@gmail.com, ORCID ID https://orcid.org/0000-0003-2070-9001. Writing the article and developing a research design.

Alexandra V. Bruter, Institute of Gene Biology

Ph.D, research fellow at the Institute of Gene Biology, Russian Academy of Sciences, e-mail: aleabruter@gmail.com, ORCID ID https://orcid.org/0000-0002-2090-2488. Developing a research design.

Diana S. Korshunova, Institute of Gene Biology

Senior technician at the Institute of Gene Biology, Russian Academy of Sciences, e-mail: korshunova@genebiology.ru, ORCID ID https://orcid.org/0000-0002-0259-7045. Coducting microinjections and working with the animals in all stages.

Leonid A. Ilchuk, Institute of Gene Biology (

Senior technician at the Institute of Gene Biology, Russian Academy of Sciences, e-mail: lechuk12@gmail.com, ORCID ID https://orcid.org/0000-0002-0157-2102. Measuring copy number and working with graphic material.

Yulia D. Okulova, Institute of Gene Biology

Senior technician at the Institute of Gene Biology, Russian Academy of Sciences, e-mail: okulova@genebiology.ru. Analyzing transgenic mice PCR analysis.

Mariya O. Soldatova, Kursk State Medical University

 2nd year student of Biology, Medical Genetics and Ecology Department, Kursk State Medical University, e-mail: mar.sold46@gmail.com, ORCID ID https://orcid.org/0000-0001-6637-1654. Genotyping

Evgeniya Seryogina, Orel State University

Research assistant of Cell Physiology and Pathology Laboratory, Orel State University, e-mail: e.s.seryogina@gmail.com, ORCID ID https://orcid.org/0000-0002-2796-4040. Writing the article.

Inga M. Kolesnik, Belgorod State National Research University

PhD in Medical Sciences, Associate Professor, Department of Pharmacology and Clinical Pharmacology, e-mail: kolesnik_i@bsu.edu.ru. The author was engaged in collection, analysis and interpretation of the data for publication.

Polina A. Ukolova, Orel State University

Research assistant of Cell Physiology and Pathology Laboratory, Orel State University, e-mail: polya.ukolova@yandex.ru, ORCID ID https://orcid.org/0000-0002-9636-513X. Writing the article.

Mikhail V. Korokin, Belgorod State National Research University

Doctor Habil. of Medical Sciences, Professor, Department of Pharmacology and Clinical Pharmacology, e-mail: mkorokin@mail.ru, ORCID ID https://orcid.org/0000-0001-5402-0697. Conceiving the idea and planning research.

Alexey V. Deykin, Institute of Gene Biology

Ph.D, senior scientist at the Institute of Gene Biology, Russian Academy of Sciences, e-mail: alexei@deikin.ru, ORCID ID https://orcid.org/0000-0001-9960-0863. Conceiving the idea and planning research.

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Опубликован

17.09.2021

Как цитировать

Kubekina MV, Silaeva YY, Bruter AV, Korshunova DS, Ilchuk LA, Okulova YD, Soldatova MO, Seryogina E, Kolesnik IM, Ukolova PA, Korokin MV, Deykin AV (2021) Transgenic mice Cre-dependently expressing mutant polymerase-gamma: novel test-system for pharmacological study of mitoprotective drugs. Research Results in Pharmacology 7(3): 33–39. https://doi.org/10.3897/rrpharmacology.7.72784

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