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Beta-lactoglobulin is the main allergen of cow’s milk. Modern approaches to reducing the allergenicity of milk require significant costs for its fermentation. An alternative approach could be the creation of productive breeds with a knockout for the gene of this protein. This will allow, at no additional cost, to increase its safety for children who are breast-fed. In this article, we report on the development of a system for gene editing to create cows with a knockout of the beta-lactoglobulin gene.

Bovine leukemia virus (BLV) causes enzootic leukemia - a chronic infectious disease occurring against the background of embedding the virus in the genome of B-lymphocytes and leading to malignization, invasion of tumor cells in organs and the formation of tumors. The disease is common in the United States, Japan, and Asia. In Russia, up to 30% is infected with BLV. Moreover, there is evidence of the presence of antibodies to the BLV virus in some groups of people, and the relationship between BLV and cancer in humans is widely discussed. All this indicates an urgent need to study BLV and create breeds resistant to it. The development of approaches to solving this problem is complicated by the fact that the receptor through which the infection is carried out is still unknown. Recently, it has been suggested that the virus penetrates the animal's lymphocytes using the CD209 molecule. In this paper, we propose a genome editing system based on CRISPR/Cas9 to get a knockout for this gene. We assume that animals obtained using the presented genome editing system will be resistant to infection with the bovine leukemia virus.

Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disorder caused by an array of mutations in the dystrophin gene, with the most commonly mutated regions being exons 48–55. One of the several existing approaches to treat DMD is gene therapy, based on alternative splicing and mutant exon skipping. Testing of such therapy requires animal models that carry mutations homologous to those found in human patients. Here, we report the generation of two genetically modified mouse lines, named "insT" and "insG", with distinct mutations at the same position in exon 51 that lead to a frameshift, presumably causing protein truncation. Hemizygous males of both lines exhibit classical signs of muscular dystrophy in all muscle tissues except for the cardiac tissue. However, pathological changes are more pronounced in one of the lines. Membrane localization of the protein is reduced to the point of absence in one of the lines. Moreover, an increase in full-length isoform mRNA was detected in diaphragms of insG line mice. Although further work is needed to qualify these mutations as sole origins of dissimilarity, both genetically modified mouse lines are suitable models of DMD and can be used to test gene therapy based on alternative splicing. [ABSTRACT FROM AUTHOR]

Inducible Cre-dependent systems are frequently used to produce both conditional knockouts and transgenic mice with regulated expression of the gene of interest. Induction can be achieved by doxycycline-dependent transcription of the wild type gene or OH-tamoxifen-dependent nuclear translocation of the chimeric Cre/ERT2 protein. However, both of these activation strategies have some limitations. We analyzed the efficiency of knockout in different tissues and found out that it correlates with the concentration of the hydroxytamoxifen and endoxifen—the active metabolites of tamoxifen—measured by LC-MS in these tissues. We also describe two cases of Cdk8floxed/floxed/Rosa-Cre-ERT2 mice tamoxifen-induced knockout limitations. In the first case, the standard scheme of tamoxifen administration does not lead to complete knockout formation in the brain or in the uterus. Tamoxifen metabolite measurements in multiple tissues were performed and it has been shown that low recombinase activity in the brain is due to the low levels of tamoxifen active metabolites. Increase of tamoxifen dosage (1.5 fold) and duration of activation (from 5 to 7 days) allowed us to significantly improve the knockout rate in the brain, but not in the uterus. In the second case, knockout induction during embryonic development was impossible due to the negative effect of tamoxifen on gestation. Although DNA editing in the embryos was achieved in some cases, the treatment led to different complications of the pregnancy in wild-type female mice. We propose to use doxycycline-induced Cre systems in such models. [ABSTRACT FROM AUTHOR]

The concept of immunological surveillance implies that immunogenic variants of tumor cells arising in the organism can be recognized by the immune system. Tumor progression is provided by somatic evolution of tumor cells under the pressure of the immune system. The loss of MHC Class I molecules on the surface of tumor cells is one of the most known outcomes of immune selection. This study developed a model of immune selection based on the immune response of TCR 1d1 single β-chain transgenic B10.D2(R101) (KdIdDb) mice to allogeneic EL4 (H-2b) thymoma cells. In wild-type B10.D2(R101) mice, immunization with EL4 cells induced a vigorous CTL response targeted to the H-2Kb molecule and results in full rejection of the tumor cells. In contrast, transgenic mice developed a compromised proliferative response in mixed-lymphocyte response assays and were unable to reject transplanted allogeneic EL4 cells. During the immune response to EL4 cells, CD8+ T-lymphocytes with endogenous β-chains accumulated predominantly in the spleen of transgenic mice and only a small part of the T-lymphocytes expressing transgenic β-chains became CD8+CD44+CD62L− effectors. Then, instead of a full elimination of tumor cells as in wild-type mice, a reproducible prolonged equilibrium phase and subsequent escape was observed in transgenic mice that resulted in death of 90% of the mice in 40-60 days after grafting. Prolonged exposure of tumor cells to the pressure of the immune system in transgenic mice in vivo resulted in a stable loss of H-2Kb molecules on the EL4 cell surface. Genetic manipulation of the T-lymphocyte repertoire was sufficient to reproduce the classic pattern of interactions between tumor cells and the immune system, usually observed in reliable syngeneic models of anti-tumor immunity. This newly-developed model could be used in further studies of immunoregulatory circuits common for transplantational and anti-tumor immune responses. [ABSTRACT FROM AUTHOR]

Atherosclerosis is a lipid-driven, chronic inflammatory disease that leads to plaque formation at specific sites of the arterial tree. Being the common cause of many cardiovascular disorders, atherosclerosis makes a tremendous impact on morbidity and mortality rates of cardiovascular diseases (CVDs) in countries with higher income. Animal models of atherosclerosis are utilized as useful tools for studying the aetiology, pathogenesis and complications of atherosclerosis, thus, providing a valuable platform for the efficacy testing of different pharmacological therapies and validation of imaging techniques. To date, a large variety of models is available. Pathophysiological changes can be induced in animals by either an atherogenic diet or genetic manipulations. The discussion of advantages and disadvantages of some murine, rabbit and porcine genetic models currently available for the atherosclerosis research is the scope of the following review., Competing Interests: None.