ISSN Print 2500–1094
ISSN Online 2542–1204
Bulletin of RSMU
BIOMEDICAL JOURNAL OF PIROGOV UNIVERSITY (MOSCOW, RUSSIA)
ORIGINAL RESEARCH
Models of mitochondrial dysfunction with inducible expression of Polg pathogenic mutant variant
About authors
1 Institute of Gene Biology, Moscow, Russia
2 Blokhin Russian Cancer Research Center, Moscow, Russia
Correspondence should be addressed: Marina V. Kubekina
Beskudnikovsky bulvar, 32, korpus 1, Moscow, 127474, Russia; moc.liamg@ymukyram
About paper
Funding: the study was supported by Russian Foundation for Basic Research, RFBR Project № 19-34-90073.
Author contribution: Kubekina MV — literature analysis, experimental research, data analysis and interpretation, oligo design, manuscript writing; Kalinina AA, Korshunova DS — experimental research; Bruter AV — literature analysis, research planning, data analysis and interpretation; Silaeva YY — literature analysis, research planning, data analysis and interpretation, scientific editing of the manuscript.
Compliance with ethical standards: the study was approved by Ethical Review board at the Institute of Gene Biology (Protocol of 05 December 2021) and carried out in strict compliance with the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.
Received: 2022-04-14
Accepted: 2022-04-28
Published online: 2022-04-27
|
Fig. 1. Genotyping bands and group-wise histograms of Polg mutant variant expression and mitochondrial membrane potential in MEF cultures. A. Genotyping for STOP-cassette (292 bp) and terminator (417 bp). B. Genotyping for CMV (100 bp) and internal control (324 bp). C. Expression of the mutant variant in Polg*Cre-CMV, Polg, CMV-Cre, and wild-type (WT) MEF cultures. Expression of the mutant variant in Polg*Cre-CMV cultures was used as a basis (=1.0) to calculate expression levels for other genotypes, and the Polg, CMV-Cre, and WT levels were averaged. D. Serial measurements of the mitochondrial membrane potential in MEF cultures over 5 weeks of culturing. The measurements for Polg, CMV-Cre, and WT control cultures were averaged and used as a basis (=1.0) to calculate the mitochondrial membrane potential for Polg*Cre-CMV cultures. * — significant differences
Fig. 2. Expression of mitophagy markers Pink1 (A), Parkin (B), Map1lc3a (C), Nix (D), and Lamp2 (E) in MEFs over 5 weeks of culturing
Fig. 3. Results of muscular endurance tests for Polg*Cre-CMV mice (dark-gray) and wild-type controls (light-gray). А. Wire hang test. B. Grip strength test. * — significant differences
Fig. 4. Decreased expression of Cd3ε, Cd3δ, and Tcr-α in mixed splenocytes of Polg*Cre-CMV mice compared with wild-type controls. * — significant differences
Fig. 5. CD4/CD8 dot plots for the single-cell suspensions of thymus cells from Polg*Cre-CMV transgenic animals (B) compared to wild-type controls (A) and a histogram of thymus cell counts (C). * — significant differences
Table. The list of primer sequences used in the study
