RESUMEN
Alzheimer's disease (AD) is the most common proteinopathy, which is accompanied by a steady decrease in the patient's cognitive functions with a simultaneous accumulation of amyloid plaques in brain tissues. Amyloid plaques are extracellular aggregates of amyloid ß (Aß) and are associated with neuroinflammation and neurodegeneration. Unlike humans and all other mammals, rats and mice do not reproduce AD-like pathology because there are three amino acid substitutions in their Aß. Amyloid plaques form in the brains of transgenic mice with overexpression of human Aß, and such mice are therefore possible to use in biomedicine to model the key features of AD. The transgenic mouse line APPswe/PS1dE9 is widely used as an animal model to study the molecular mechanisms of AD. A study was made to characterize the APPswe/PS1dE9/Blg subline, which was obtained by crossing APPswe/PS1dE9 mice on a CH3 genetic background with C57Bl6/Chg mice. No difference in offspring's survival and fertility was observed in the subline compared to wild-type control mice. Histological analysis of the brain in the APPswe/PS1dE9/Blg line confirmed the main neuromorphological features of AD and showed that amyloid plaques progressively increase in number and size during aging. The APPswe/PS1dE9/Blg line was assumed to provide a convenient model for developing therapeutic strategies to slow down AD progression.
Asunto(s)
Enfermedad de Alzheimer , Angiopatía Amiloide Cerebral , Ratones , Humanos , Ratas , Animales , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Ratones Transgénicos , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Placa Amiloide/genética , Angiopatía Amiloide Cerebral/genética , Angiopatía Amiloide Cerebral/complicaciones , Angiopatía Amiloide Cerebral/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , MamíferosRESUMEN
The peripheral T-cell pool consists of several, functionally distinct populations of CD8+ T cells. CD44 and CD62L are among the major surface markers that allow us to define T-cell populations. The expression of these molecules depends on the functional status of a T lymphocyte. Under lymphopenic conditions, peripheral T cells undergo homeostatic proliferation and acquire the memory-like surface phenotype CD44hiCD62Lhi. However, the data on the functional activity of these cells remains controversial. In this paper, we analyzed the effects of the adoptive transfer of syngeneic splenocytes on the recovery of CD8+ T cells in sublethally irradiated mice. Our data demonstrate that under lymphopenia, donor lymphocytes form a population of memory-like CD8+ T cells with the phenotype CD122+CD5+CD49dhiCXCR3+ that shares the phenotypic characteristics of true memory cells and suppressive CD8+ T cells. Ex vivo experiments showed that after adoptive transfer in irradiated mice, T cells lacked the functions of true effector or memory cells; the allogeneic immune response and immune response to pathogens were greatly suppressed in these mice.
RESUMEN
Under conditions of lymphopenia, T lymphocytes proliferate and acquire a surface activation phenotype, which in many respects is similar to the phenotype of true memory T cells. We investigated the phenotypic features of the CD8+ T-cell population formed from donor lymphocytes after adoptive transfer of syngeneic splenocytes to sublethally irradiated mice. This population expresses markers CD44, CD122, CD5, CD49d and the chemokine receptor CXCR3. Thus, for the first time, the phenomenon of the formation of a population of T cells with signs of suppressive CD8+ T lymphocytes and true memory cells was demonstrated.
Asunto(s)
Traslado Adoptivo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Linfopenia/inmunología , Bazo/inmunología , Animales , Biomarcadores/metabolismo , Recuento de Células , Regulación de la Expresión Génica/inmunología , Ratones , FenotipoRESUMEN
Atherosclerosis retains the leading position among the causes of global morbidity and mortality worldwide, especially in the industrialized countries. Despite the continuing efforts to investigate disease pathogenesis and find the potential points of effective therapeutic intervention, our understanding of atherosclerosis mechanisms remains limited. This is partly due to the multifactorial nature of the disease pathogenesis, when several factors so different as altered lipid metabolism, increased oxidative stress, and chronic inflammation act together leading to the formation and progression of atherosclerotic plaques. Adequate animal models are currently indispensable for studying these processes and searching for novel therapies. Animal models based on rodents, such as mice and rats, and rabbits represent important tools for studying atherosclerosis. Currently, genetically modified animals allow for previously unknown possibilities in modelling the disease and its most relevant aspects. In this review, we describe the recent progress made in creating such models and discuss the most important findings obtained with them to date.
Asunto(s)
Aterosclerosis , Modelos Animales de Enfermedad , Animales , Animales Modificados Genéticamente , Aterosclerosis/fisiopatología , Progresión de la Enfermedad , Humanos , Ratones , Conejos , RatasRESUMEN
CDK8-mediated transcriptional reprogramming is essential for an extensive gene expression. Constitutive knockouts of the cdk8 gene are lethal at the morula stage. For modeling transcriptional reprogramming in an adult organism, we investigated the possibility to attenuate the CDK8 kinase activity with a F97G mutation in exon 3 of the cdk8 gene. According to preliminary experimental data, this mutation should lead to a decrease in CDK8 kinase activity. To edit the genome of laboratory mice, the CRISPR/Cas9 technology was used, in which the introduction of a double-stranded gap occurred at a distance of 128 nucleotide pairs from the planned site of the introduced mutation. To introduce the mutation, a matrix for homologous repair was used as part of plasmid DNA, with homologous arms 903 and 484 bp in the 5'-3' region from the point of double-stranded rupture, respectively. As a result, mice with site-specific target mutations in exon 3 of the cdk8 gene were obtained. We for the first time demonstrated a high efficacy of the mutation 128 bp apart from the site of double-strand break. Viable animals with the F97G mutation in the catalytic domain of CDK8 kinase were obtained for the first time. The resulting cdk8 mutant mice will be used in subsequent studies to simulate the processes involving transcription reprogramming.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/metabolismo , Edición Génica/métodos , Genoma , ARN Guía de Kinetoplastida , Transcripción Genética , Animales , Sistemas CRISPR-Cas , Dominio Catalítico , Exones , Heterocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Mutación , Oligonucleótidos/genéticaRESUMEN
Atherosclerosis retains the leading position among the causes of global morbidity and mortality worldwide, especially in the industrialized countries. Despite the continuing efforts to investigate disease pathogenesis and find the potential points of effective therapeutic intervention, our understanding of atherosclerosis mechanisms remains limited. This is partly due to the multifactorial nature of the disease pathogenesis, when several factors so different as altered lipid metabolism, increased oxidative stress, and chronic inflammation act together leading to the formation and progression of atherosclerotic plaques. Adequate animal models are currently indispensable for studying these processes and searching for novel therapies. Animal models based on rodents, such as mice and rats, and rabbits represent important tools for studying atherosclerosis. Currently, genetically modified animals allow for previously unknown possibilities in modelling the disease and its most relevant aspects. In this review, we describe the recent progress made in creating such models and discuss the most important findings obtained with them to date.
Asunto(s)
Humanos , Animales , Ratones , Conejos , Ratas , Modelos Animales de Enfermedad , Aterosclerosis/fisiopatología , Animales Modificados Genéticamente , Progresión de la EnfermedadRESUMEN
A multiplex PCR test system for identification of the regulatory sequences of genetic constructs for transformation (promotor, insulator, and terminator) in the Mus musculus genome and for transgenic animal selection by genotyping with horizontal agarose gel electrophoresis detection was developed. The proposed system was validated by genotyping mouse strains producing human lactoferrin, heat shock protein HSP 70, firefly luciferase, and lysozyme, which were obtained by microinjections of linearized DNA into murine zygote pronucleus with random transgene integration into the genome using the pBC1 plasmid for expression of the gene of interest in milk of transformed animals (milk expression vector kit).
Asunto(s)
Técnicas de Genotipaje , Lactoferrina , Leche/metabolismo , Reacción en Cadena de la Polimerasa Multiplex , Animales , Lactoferrina/genética , Lactoferrina/metabolismo , Ratones , Ratones TransgénicosRESUMEN
Animal models of diseases are invaluable tools of modern medicine. More than forty years have passed since the first successful experiments and the spectrum of available models, as well as the list of methods for creating them, have expanded dramatically. The major step forward in creating specific disease models was the development of gene editing techniques, which allowed for targeted modification of the animal's genome. In this review, we discuss the available tools for creating transgenic animal models, such as transgenesis methods, recombinases, and nucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems. We then focus specifically on the models of atherosclerosis, especially mouse models that greatly contributed to improving our understanding of the disease pathogenesis and we outline their characteristics and limitations.
Asunto(s)
Animales Modificados Genéticamente , Aterosclerosis/fisiopatología , Modelos Animales de Enfermedad , Ingeniería Genética/métodos , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Animales , Aterosclerosis/genética , Investigación Biomédica/métodos , Femenino , Técnicas de Transferencia de Gen , Humanos , Masculino , RatonesRESUMEN
Animal models of diseases are invaluable tools of modern medicine. More than forty years have passed since the first successful experiments and the spectrum of available models, as well as the list of methods for creating them, have expanded dramatically. The major step forward in creating specific disease models was the development of gene editing techniques, which allowed for targeted modification of the animal's genome. In this review, we discuss the available tools for creating transgenic animal models, such as transgenesis methods, recombinases, and nucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems. We then focus specifically on the models of atherosclerosis, especially mouse models that greatly contributed to improving our understanding of the disease pathogenesis and we outline their characteristics and limitations.
Asunto(s)
Humanos , Animales , Masculino , Femenino , Conejos , Animales Modificados Genéticamente , Ingeniería Genética/métodos , Modelos Animales de Enfermedad , Aterosclerosis/fisiopatología , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Técnicas de Transferencia de Gen , Investigación Biomédica/métodos , Aterosclerosis/genéticaRESUMEN
The use of transgenic animals as bioreactors for the synthesis of the recombinant proteins secreted into milk is a current trend in the development of biotechnologies. Advances in genetic engineering, in particular the emergence of targeted genome editing technologies, have provided new opportunities and significantly improved efficiency in the generation of animals that produce recombinant proteins in milk, including economically important animals. Here, we present a retrospective review of technologies for generating transgenic animals, with emphasis on the creation of animals that produce recombinant proteins in milk. The current state and prospects for the development of this area of biotechnology are discussed in relation to the emergence of novel genome editing technologies. Experimental and practical techniques are briefly discussed.
RESUMEN
AIM: To evaluate an effect of dimebon on the onset of symptomatic stage in FUS.1-513 transgenic mice - a new genetic model of neurodegeneration, and to study the dynamics of disease progression in the terminal stage. MATERIAL AND METHODS: The study was carried out on males of line FUS1-513 with the contribution of genes from CD1 strains. Mice of the experimental group (n=28) received dimebon with water in the concentration of 70 mcg/ml starting from the 35th day of life. The control group (n=25) did not receive the drug. Age, body mass of animals at the start of symptomatic stage and duration of symptomatic stage were assessed. RESULTS: Application of dimebon can delay the onset of the manifestation of clinical symptoms of the neurodegenerative process in the experimental group (127.6±4.6 days) compared to the control group (110.6±4.2 days). The body mass was similar in both groups. CONCLUSION: Dimebon leads to an increase in the duration of presymptomatic stage and delays the manifestation of clinical symptoms. The changes in the dynamics of the pathological process in the symptomatic stage are not detected.
Asunto(s)
Esclerosis Amiotrófica Lateral , Indoles , Esclerosis Amiotrófica Lateral/prevención & control , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Indoles/uso terapéutico , Masculino , Ratones , Ratones TransgénicosRESUMEN
During the past two decades, there have been numerous attempts at using animals in order to produce recombinant human proteins and monoclonal antibodies. However, it is only recently that the first two therapeutic agents isolated from the milk of transgenic animals, C1 inhibitor (Ruconest) and antithrombin (ATryn), appeared on the market. This inspires hope that a considerable number of new recombinant proteins created using such technology could become available for practical use in the near future. In this review, the methods applied to produce transgenic animals are described and the advantages and drawbacks related to their use for producing recombinant human proteins and monoclonal antibodies are discussed.
RESUMEN
Genetic constructs containing the human lactoferrin (hLf) gene were created within a joint program of Russian and Belorussian scientists. Using these constructs, transgenic mice were bred (the maximum hLf concentration in their milk was 160 g/L), and transgenic goats were also generated (up to 10 g/L hLf in their milk). Experimental goatherds that produced hLf in their milk were also bred, and the recombinant hLf was found to be identical to the natural protein in its physical and chemical properties. These properties included electrophoretic mobility, isoelectric point, recognition by polyclonal and monoclonal antibodies, circular dichroic spectra, interaction with natural ligands (DNA, lipopolysaccharides, and heparin), the binding of iron ions, the sequence of the 7 terminal amino acids, and its biological activity. The latter was assessed by the agglutination of Micrococcus luteus protoplasts, bactericidal activity against Escherichia coli and Listeria monocytogenes , and fungicidal activity against Candida albicans . We also demonstrated a significant increase in the activity of antibiotics when used in combination with Lf.