Creation of Cybrid Cultures Containing mtDNA Mutations m.12315G>A and m.1555G>A, Associated with Atherosclerosis.

Margarita A Sazonova,Vasily P Karagodin,Tatiana P Shkurat,Alexander N Orekhov,Igor A Sobenin,Marina D Sazonova,Anastasia I Ryzhkova,Zukhra B Khasanova,Vasily V Sinyov

doi:10.3390/biom9090499

Abstract

In the present work, a pilot creation of four cybrid cultures with high heteroplasmy level was performed using mitochondrial genome mutations m.12315G>A and m.1555G>A. According to data of our preliminary studies, the threshold heteroplasmy level of mutation m.12315G>A is associated with atherosclerosis. At the same time, for a mutation m.1555G>A, such a heteroplasmy level is associated with the absence of atherosclerosis. Cybrid cultures were created by fusion of rho0-cells and mitochondria from platelets with a high heteroplasmy level of the investigated mutations. To create rho0-cells, THP-1 culture of monocytic origin was taken. According to the results of the study, two cybrid cell lines containing mutation m.12315G>A with the heteroplasmy level above the threshold value (25% and 44%, respectively) were obtained. In addition, two cybrid cell lines containing mutation m.1555G>A with a high heteroplasmy level (24%) were obtained. Cybrid cultures with mtDNA mutation m.12315G>A can be used to model both the occurrence and development of atherosclerosis in cells and the titration of drug therapy for patients with atherosclerosis. With the help of cybrid cultures containing single nucleotide replacement of mitochondrial genome m.1555G>A, it is possible to develop approaches to the gene therapy of atherosclerosis.

Highlights

At present, cybrid cell cultures are the most promising models for studying pathological processes in cells in various diseases
It should be noted that for the creation of morphologically homogeneous and resistant to repeated passaging of the cybrid lines, in general, established cell lines are used, i.e., those cells which have passed the stage of dedifferentiation
The absence of mitochondria in the culture was confirmed by the analysis of the number of mitochondrial genome copies in rho0 culture using the real-time PCR method

Summary

Introduction

Cybrid cell cultures are the most promising models for studying pathological processes in cells in various diseases They may be used for studying the impact of mtDNA variants on the occurrence of mitochondrial dysfunction and different pathologies in humans [1,2,3,4,5]. One of the relevant goals of medicine and public health is the creation of cellular models containing pathogenic mutations for the development of drug therapy of various pathologies. The advanced field of molecular medicine encompasses the creation of cybrid cell cultures containing mutations with an anti-pathological effect. These cultures are necessary for gene therapy of human diseases. As a result of mutation m.1555G>A, the 12S subunit of ribosomal RNA can stabilize, leading to an acceleration of the protein chains synthesis on the mitochondrial ribosome

Results and Discussion

Creation of rho0 Cell Cultures

Isolation

Conclusions