Generation of two iPSC lines from patient with Mucopolysaccharidosis IV B type and autosomal recessive non-syndromic hearing loss 12.

We generated two human induced pluripotency stem cell (hiPSC) lines, RCMGi011-A and 11-B, from skin fibroblast from patient with Mucopolysaccharidosis IV B type and autosomal recessive non-syndromic hearing loss 12 using non-integrating, viral CytoTune™-iPS 2.0 Sendai Reprogramming Kit. We verified variant c.808 T > G and insertion in GLB1 gene, as well as two mutations, c.6992 T > C and c.805C > T, in CDH23 gene which lead to autosomal recessive hearing loss type 12. We have demonstrated normal karyotype of hiPSCs and capacity for cell differentiation into three germ layers.

[The Choice of a Donor Molecule in Genome Editing Experiments in Animal Cells].

Genome editing is a powerful tool that allows study of the properties of genes or changes to be made to the genetic sequence. Programmable nucleases that can induce double-strand breaks in the genomic sequence of interest have been developed over the past few decades. After initiation of a double-strand break (DSB) in DNA, the DSB can be repaired by the NHEJ (non-homologous end joining), which leads to various errors and gene knockout. Other repair options, HDR (homology directed repair) or SSTR (single-strand template repair), allow researchers to make desired changes in the gene. HDR occurs in the presence of a donor template, in natural conditions the donor template is a sister chromatid. The efficiency of HDR and SSTR is significantly lower than the efficiency of NHEJ in genome editing. Double-stranded, single-stranded and long single-stranded DNAs are used to increase efficiency and to make desired changes in genomic DNA. In this review, we discuss donor molecules that are used for DSB repair using HDR or SSTR during genome editing, their application, and modifications to increase the efficiency of HDR and SSTR.

Animal models for researching approaches to therapy of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a relatively widespread genetic disease which develops as a result of a mutation in the gene DMD encoding dystrophin. In this review, animal models of DMD are described. These models are used in preclinical studies to elucidate the pathogenesis of the disease or to develop effective treatments; each animal model has its own advantages and disadvantages. For instance, Caenorhabditis elegans, Drosophila melanogaster, and zebrafish (sapje) are suitable for large-scale chemical screening of large numbers of small molecules, but their disease phenotype differs from that of mammals. The use of larger animals is important for understanding of the potential efficacy of various treatments for DMD. While mdx mice have their advantages, they exhibit a milder disease phenotype compared to humans or dogs, making it difficult to evaluate the efficacy of new treatment for DMD. The disease in dogs and pigs is more severe and progresses faster than in mice, but it is more difficult to breed and obtain sufficient numbers of specimens in order to achieve statistically significant results. Moreover, working with large animals is also more labor-intensive. Therefore, when choosing the optimal animal model for research, it is worth considering all the goals and objectives.

TIRR: a potential front runner in HDR race-hypotheses and perspectives.

The majority of CRISPR-Cas9 methods for mutations correction are oriented on gene editing through homologous recombination that is normally restrained by non-homologous end joining (NHEJ). A recently identified protein TIRR can bind a 53BP1 protein, a key effector of NHEJ, and inhibit its recruitment to double-strand break loci. Several studies elucidated the molecular mechanisms of TIRR-53BP1 binding and established bidirectional role of TIRR in 53BP1 functions and stability. It was proved that overexpression of TIRR promotes the double-strand break repair through homologous recombination. All findings, which were described in the review, allow assuming TIRR as a suitable target for enhancing efficacy of genome editing through homology directed repair.

Dioxidine-induced changes in genome-wide DNA methylation in a culture of peripheral blood lymphocytes.

We studied the effect of dioxidine on genome-wide methylation in short-term cultures of peripheral blood lymphocytes derived from healthy donors. Methylation was evaluated in lymphocytes before culturing, after 25 h in culture, and 1 h after addition of dioxidine in two concentrations (0.1 and 0.01 mg/ml). The total time in culture was 25 h. The level of methylation was assessed using methyl-sensitive single-cell gel electrophoresis ("comet assay") with additional restriction with HpaII amd MspI. Significant individual differences were found in the levels of methylation in both native cells and in cells treated with dioxidine in both concentrations. Mean group indicators of methylation did not differ before culturing and after 25 h in culture (45.28 and 44.80%, respectively). The mean group rate of methylation increased to 46.14% (p<0.001) after dioxidine treatment in a concentration of 0.01 mg/ml. Dioxidine in 0.1 mg/ml reduced the level of methylation (mean group rate 42.31%; p<0.001).

[Mutagen influence with different mechanisms of action on DNA global methylation in human whole-blood lymphocytes in vitro].

Data that support the evidence of mutagens known to cause epigenetic abnormalities that could potentially result in genomic instability and the development of cancer rather than to modifications in the human genome at the gene and chromosomal levels only. The level of global methylation in human lymphocytes in vitro caused by exposure to two mutagens with different mechanisms of action, i.e., dioxidine and methyl methanesulphonate (MMS), was demonstrated in the present study. Global methylation was assessed by methyl-sensitive comet assay. An increase in the level of global methylation to 45.64% was revealed during culturing with dioxidine in a concentration of 0.01 mg/mL (p < 0.001), while the addition of dioxidine in a concentration of 0.1 mg/mL resulted in a decreased level of methylation up to 42.31% (p < 0.001). The addition of M MS in concentrations of 0.0025 and 0.01 mg/mL resulted in minor but significant modifications (p < 0.05) of the global methylation level ranged within natural variations in global methylation. Accordingly, the addition ofdioxidine in the concentration of 0.1 mg/mL might cause genomic instability and might be considered a potential carcinogen.

[VEGF gene expression in transfected human multipotent stromal cells].

Dynamics of VEGF gene expression in transfected multipotent stromal cells from adipose tissue was examined using electroporation and lipofection. Differences in the potency and dynamics of plasmid elimination (up to day 9) between cell cultures were observed. All cultures were divided into fast and slow plasmid-eliminating ones. Interculture differences in VEGF expression were detected. The possibility of a 5-6-fold increase of VEGF expression was shown. There were no differences in transfection potency, plasmid elimination dynamics, and VEGF expression after transfection by both nonviral methods.

Dynamics of elimination of plasmids and expression of VEGF121 gene transfected into human mesenchymal stem cells by different methods.

We compared two methods of transfection (lipofection and electroporation) with plasmid containing VEGF121 gene in four cultures of mesenchymal stem cells from the human adipose tissue. The efficacy of transfection after 1 day, the dynamics of plasmid elimination after 3, 6, 9 days, and expression of the target gene were evaluated. Transfection by both methods failed in one of 4 cultures. Analysis of the plasmid elimination dynamics showed that the content of plasmids introduced by both methods decreased by 30-69% in all cultures by day 3 and then remained unchanged from day 3 to day 9. The expression of the target gene did not correlate with the content of plasmids in cells and varied by 2-10 times in control cells and cells transfected by both methods. Fluctuation of VEGF121 expression was not related to methylation.

[Methods for detection of methylated cytosine residues in DNA].

The article provides analysis of common methods for DNA methylation detection. Advantages and limitations of methods used for different purposes are compared. Clue step for most common methods is bisulfite treatment of DNA samples and its protocol is described in details. Recommendations are formulated for each method best in solving specific problems.

[The clinical pharmacology of beta-adrenoblockers].

Beta-adrenoblockers (BAB) are highly-effective pharmaceuticals used broadly in treatment of cardial diseases. Response to BABs is known to display interindividual variability; pre-treatment analysis of allelic gene variants responsible for BAB pharmacokinetics, allows dose correction according to the genetic features of an individual patient. Pharmacogenetics is a young science, but it has already proven its practical significance in term of individualization of pharmacotherapy, which in some cases makes it possible to increase the effectiveness of the pharmaceuticals and avoid undesirable effects.