Genetically modified mice as a tool for the study of human diseases.

Modeling a human disease is an essential part of biomedical research. The recent advances in the field of molecular genetics made it possible to obtain genetically modified animals for the study of various diseases. Not only monogenic disorders but also chromosomal and multifactorial disorders can be mimicked in lab animals due to genetic modification. Even human infectious diseases can be studied in genetically modified animals. An animal model of a disease enables the tracking of its pathogenesis and, more importantly, to test new therapies. In the first part of this paper, we review the most common DNA modification technologies and provide key ideas on specific technology choices according to the task at hand. In the second part, we focus on the application of genetically modified mice in studying human diseases.

Cyclophilin A as a Pro-Inflammatory Factor Exhibits Embryotoxic and Teratogenic Effects during Fetal Organogenesis.

The precise balance of Th1, Th2, and Th17 cytokines is a key factor in successful pregnancy and normal embryonic development. However, to date, not all humoral factors that regulate and influence physiological pregnancy have been completely studied. Our data here pointed out cyclophilin A (CypA) as the adverse pro-inflammatory factor negatively affecting fetal development and associated with pregnancy complications. In different mouse models in vivo, we demonstrated dramatic embryotoxicity and teratogenicity of increased CypA levels during pregnancy. Using generated transgenic models, we showed that CypA overexpression in fetal tissues induced the death of all transgenic fetuses and complete miscarriage. Administration of recombinant human CypA in a high dose to pregnant females during fetal organogenesis (6.5-11.5 dpc) exhibited teratogenic effects, causing severe defects in the brain and bone development that could lead to malformations and postnatal behavioral and cognitive disorders in the offspring. Embryotoxic and teratogenic effects could be mediated by CypA-induced up-regulation of M1 macrophage polarization via activation of the STAT1/3 signaling pathways. Here, we propose secreted CypA as a novel marker of complicated pregnancy and a therapeutic target for the correction of pregnancy complications.

Genetically Engineered Mice Unveil In Vivo Roles of the Mediator Complex.

The Mediator complex is a multi-subunit protein complex which plays a significant role in the regulation of eukaryotic gene transcription. It provides a platform for the interaction of transcriptional factors and RNA polymerase II, thus coupling external and internal stimuli with transcriptional programs. Molecular mechanisms underlying Mediator functioning are intensively studied, although most often using simple models such as tumor cell lines and yeast. Transgenic mouse models are required to study the role of Mediator components in physiological processes, disease, and development. As constitutive knockouts of most of the Mediator protein coding genes are embryonically lethal, conditional knockouts and corresponding activator strains are needed for these studies. Recently, they have become more easily available with the development of modern genetic engineering techniques. Here, we review existing mouse models for studying the Mediator, and data obtained in corresponding experiments.

Physiological and Functional Effects of Dominant Active TCRα Expression in Transgenic Mice.

A T cell receptor (TCR) consists of α- and ß-chains. Accumulating evidence suggests that some TCRs possess chain centricity, i.e., either of the hemi-chains can dominate in antigen recognition and dictate the TCR's specificity. The introduction of TCRα/ß into naive lymphocytes generates antigen-specific T cells that are ready to perform their functions. Transgenesis of the dominant active TCRα creates transgenic animals with improved anti-tumor immune control, and adoptive immunotherapy with TCRα-transduced T cells provides resistance to infections. However, the potential detrimental effects of the dominant hemi-chain TCR's expression in transgenic animals have not been well investigated. Here, we analyzed, in detail, the functional status of the immune system of recently generated 1D1a transgenic mice expressing the dominant active TCRα specific to the H2-Kb molecule. In their age dynamics, neither autoimmunity due to the random pairing of transgenic TCRα with endogenous TCRß variants nor significant disturbances in systemic homeostasis were detected in these mice. Although the specific immune response was considerably enhanced in 1D1a mice, responses to third-party alloantigens were not compromised, indicating that the expression of dominant active TCRα did not limit immune reactivity in transgenic mice. Our data suggest that TCRα transgene expression could delay thymic involution and maintain TCRß repertoire diversity in old transgenic mice. The detected changes in the systemic homeostasis in 1D1a transgenic mice, which are minor and primarily transient, may indicate variations in the ontogeny of wild-type and transgenic mouse lines.

The mitochondrial challenge: Disorders and prevention strategies.

This short review provides basic knowledge on mitochondrial inheritance, its disorders, and potential ways to overcome them in human reproductive medicine. The latter are currently mostly associated with the so-called mitochondrial replacement (nuclear transfer) procedures, performed at different stages and with slight technical differences. Being promising but obviously highly invasive, these procedures require detailed investigation of their delayed effects on embryogenesis, pregnancy and future health. A special attention is paid to the newest available data on these issues, as well as to their limitations and possible further research directions.

Limitations of Tamoxifen Application for In Vivo Genome Editing Using Cre/ERT2 System.

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.

Femtosecond laser oocyte enucleation as a low-invasive and effective method of recipient cytoplast preparation.

Recipient cytoplast preparation, commonly performed by DNA aspiration with a needle, inevitably leads to the loss of reprogramming factors. As an alternative to the traditional enucleation technique, femtosecond laser enucleation can eliminate DNA effectively without loss of reprogramming factors and without oocyte puncturing. In this work we have performed oocyte enucleation by destructing the metaphase plate using a 795 nm femtosecond laser. The disability of the enucleated oocytes to develop after the parthenogenetic activation, as well as the lack of DNA staining luminescence, strongly confirms the efficiency of the femtosecond laser enucleation. The parthenogenetic development of oocytes after the cytoplasm treatment suggests a low-invasive effect of the laser enucleation technique.

Safety evaluation of the mouse TCRα - transduced T cell product in preclinical models in vivo and in vitro.

Adoptive cell therapy (ACT) based on TCR- or CAR-T cells has become an efficient immunotherapeutic approach for the treatment of various diseases, including cancer. Previously, we developed a novel strategy for generating therapeutic T cell products based on chain-centric TCRs, in which either α- or ß-chain dominates in cognate antigen recognition. To assess the suitability of our experimental approach for the clinical application and predict its possible adverse effects, in studies here, we evaluated the safety of the experimental TCRα-modified T cell product in mouse preclinical models. Our data showed no tumorigenic or mutagenic activity in vitro of TCRα-transduced T cells, indicating no genotoxicity of viral vectors used for the generation of the experimental T cell product. Adoptive transfer of TCRα-engineered T cells in a wide dose range didn`t disturb the host homeostasis and exhibited no acute toxicity or immunotoxicity in vivo. Based on pharmacokinetics and pharmacodynamics analysis here, modified T cells rapidly penetrated and distributed in many viscera after infusion. Histological evaluations revealed no pathological changes in organs caused by T cells accumulation, indicating the absence of non-specific off-target activity or cross-reactivity of the therapeutic TCRα. Studies here provide valuable information on the potential safety of TCRα-T cell based ACT that could be extrapolated to possible effects in a human host.

Modelling of atherosclerosis in genetically modified animals.

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.

Immune selection of tumor cells in TCR ß-chain transgenic mice.

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 ß-chain transgenic B10.D2(R101) (K(d)I(d)D(b)) mice to allogeneic EL4 (H-2(b)) thymoma cells. In wild-type B10.D2(R101) mice, immunization with EL4 cells induced a vigorous CTL response targeted to the H-2K(b) 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 ß-chains accumulated predominantly in the spleen of transgenic mice and only a small part of the T-lymphocytes expressing transgenic ß-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-2K(b) 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.