Several humanized transgenic lines of biomodel mice containing an integrated variable human gene of the main histocompatibility complex (MHC) have been created at the Federal State Budgetary Scientific and Scientific Research Institute of the FMBA of Russia. These include HLA-A*02:01, HLA-B*07:02 and HLA-C*07:02. The lines were created by microinjection of a linear fragment of a genetically engineered structure (GES) into the male pronucleus of zygotes, followed by the transfer of potentially modified embryos into the reproductive tract to pseudo-pregnant female recipients. The created GES encodes a chimeric molecule of the MHC of class I on the cell surface, consisting of the α1-, α2-domains of human HLA, and the α3-domain of the mouse H-2K complex, stabilized by human β2-microglobulin connected by a glycine serine linker with the α1-domain of HLA [1–5]. The created biomodels can be successfully used to solve a wide range of research tasks, including studies of immune reactions, infectious, autoimmune and oncological diseases, as well as the development and testing of vaccines in the field of pharmacosafety and immunogenicity. This article presents theoretical information on the genetic polymorphism of the studied gene in the human genome, as well as experimental data on the transgenic lines of biomodels created by the authors and the results of comparing the allele-specific site in the obtained animal lines. The analysis was performed by Sanger sequencing on a cDNA matrix.

Transgenic humanized animals are increasingly in demand for biomedical research and pharmacological testing. More and more lines of transgenic animals are being created, including those with knockout of their own genes. There is an urgent need for an evidence base for the integration of a transgene, its expression, determination of the knockout state of its own gene at the molecular genetic level, detection of translation of the target protein in different organs and tissues, proof for the absence of protein synthesis (or its non-functionality), the gene of which has been modified. This requires highly specific reagents, proteins and antibodies to them in particular, the vast majority of which are presented by foreign manufacturers. The task was set to identify mouse and human β2-microglobulin in protein fractions of organs and tissues of transgenic and knockout mice of several HLA lines created in recent years at the Scientific Center of Biomedical Technologies, Russia. At the first stage of our research, recombinant E. coli producing strains were obtained.

Human leukocyte antigen plays a primary role in the formation of immune response and pathogenesis of   diseases of various etiologies, including the development of negative side effects induced by pharmacological agents. Modern pharmacosafety standards require improvement of existing test systems to conduct high-quality preclinical studies. A number of humanized transgenic mouse lines with hybrid HLA I class molecules on the cell surface, which correspond to the human allelic variants HLA-A*02:01, HLA-B*07:02, and  HLA-C*07:02, were developed at the Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia. In this article, we present experimental data on quantitative determination of β2-microglobulin protein and HLA by the “sandwich” ELISA method in mice with different alleles of   HLA I class genes. The results obtained confirm the presence of target functional proteins (transgenicity) in humanized transgenic mice, which is consistent with our previous data obtained when determining the primary sequence of the transgene using Sanger sequencing. We also discuss the scientific and practical significance of such biomodels, as well as the scope of their application.

The introduction of a transgene can impact negatively the functioning of vital systems in biomodels. We  carried out a comparative analysis of the immune response of mice of the HLA-A*02:01 humanized transgenic line, mice with mouse β2-microglobulin gene knockout, and wild-type mice to the introduction of horse immunoglobulin as an antigen. The biomodel lines were created at the Scientific Center of Biomedical Technologies of the Federal Medical and Biological Agency of Russia. The maximum immune response was achieved on the 30th day from the onset of immunization in animals of the HLA-A*02:01 line and wild-type mice. Antibody titers in these groups increased sharply and approached 1:8,000,000 and 1:4,000,000, respectively. This indicates that genome modification in HLA-A*02:01 transgenic humanized mice did not affect functioning of the immune system. No similar dynamics of the increase in antibody titers was observed in the mice line with mouse β2-microglobulin gene knockout. On the 7th and 30th day, the antibody titer in this group increased to a value of 1:400 and 1:6,400, respectively. The weak immune response in mice with mouse β2-microglobulin gene knockout confirms the undeniably important role of  this protein in immune response formation.

Highly specific reagents, i.e., proteins and antibodies to them, are the necessary components of systems for verifying the effectiveness of transgenic/knockout animal biomodels. In particular, the identification of   mouse and human β2-microglobulin in the protein fractions of organs and tissues of transgenic and β2m knockout mice of several HLA lines, which have been created in recent years at the Scientific Center of  Biomedical Technologies of the FMBA of Russia, is the most important stage of their certification. At  the first stage of our research,  E. coli  producing strains of recombinant mouse and human β2-microglobulin (mβ2mg and hβ2mg) were obtained, the proteins were isolated and purified. At the next stage of the work, affine sorbents with immobilized mβ2mg and hβ2mg were obtained. To increase the species specificity of the serum, “rabbit-anti-hβ2mg” were depleted against the recombinant protein mβ2mg, and, conversely, “rabbit-anti-mβ2mg” were depleted against the recombinant protein hβ2mg. Highly specific antibodies were purified from depleted sera using affinity sorbents. Using dot- and western-blotting methods оn the example of depleted and affinity-purified rabbit-anti-hβ2mg antibodies, a significant increase in  their specificity relative to hβ2mg was shown.

A systematic study of γ-oscillations was carried out using rats with chronically implanted electrodes in the proreal gyrus, somatosensory cortex, dorsal hippocampus, and hypothalamus. Brain electrograms (BE) were recorded and investigated using an original software and hardware module. Linear diagrams were constructed using a QMS17 device in a frequency range of 60–250 Hz or greater. A mathematical analysis, normalization, and rationing of the series of γ-rhythms under the action of gamma-aminobutyric acid (GABA), acetylcholine (ACC), and insulin relative to similar background series were performed by double discrete-time Fourier transform and double angle arctangent function, which allowed us to extract relevant information from extremely small (1–2 μV) values of γ-oscillations. The accumulation of the substances under study was achieved by introducing the Aminalon (GABA), Galantamine (ACC), and liposomal Insulin pharmaceuticals. The plasma concentrations of the studied drugs were verified by HPLC and mathematical modeling. The normalized BE (NBE) reflected the intracentral mechanisms of action of the tested drugs, which were characterized by a stable picture in the resting state of the animals and under the action of Aminalon, Galantamine, and Insulin at the peak of their plasma concentrations (according to pharmacokinetic parameters). The γ-activity of the brain is maintained at the systemic level. Blockade of γ-oscillations in the frontal pole leads to their activation in the associated brain structures: the hypo-thalamus, reticular formation, caudate nucleus, etc. Under the influence of Aminalon, the total depressive effects were observed over the entire analyzed range in the posterior nucleus of the hypothalamus and proreal gyrus, as well as activating effects in the frequency range 60–75 Hz in the anterior suprasylvian gyrus. Under the action of Galantamine, partial depressive effects in the hippocampus and hypothalamus were observed at   frequencies of about 60–65, 95–105, and 150 Hz. Under the action of liposomal Insulin, partial activating effects were noted in the anterior suprasylvian gyrus and in the dorsal hippocampus in the frequency range of 60–85 Hz.

This article presents the results of 10-year research studies conducted using minipigs at the Scientific Center of Biomedical Technologies. Comparisons with the most significant laboratory animals are presented. Prospects for involving minipigs in various biomedical manipulations as an alternative to monkeys, whose use is restricted, are shown.

In this work, we investigate the pharmacokinetics of a new anti-inflammatory hexapeptide registered under the name of Leutragin. The study was conducted on Svetlogorsk minipigs by intravenous and a single rectal administration of the drug in the form of a solution and suppositories at an equal dose of 10 mg. The shortest time to reach peak concentration was demonstrated with intravenous administration, with the Tmax being 30 min. The maximum concentration (Cmax) when administering Leutragin in a suppository form was 141.37 ng/g. This concentration was achieved at the Tmax of 90 min, following which Leutragin remained in the bloodstream for 2.5 h. The absolute bioavailability of Leutragin in the suppository and solution form was 59.6% and 70.03%, respectively. The peak concentration of Leutragin under its rectal administration occurred at 150 min, following with the drug remained in the bloodstream for 4 h.