Decrease in pool of T lymphocytes with surface phenotypes of effector and central memory cells under influence of TCR transgenic ß-chain expression.

Peripheral T lymphocytes can be subdivided into naïve and antigen-experienced T cells. The latter, in turn, are represented by effector and central memory cells that are identified by different profiles of activation markers expression, such as CD44 and CD62L in mice. These markers determine different traffic of T lymphocytes in the organism, but hardly reproduce real antigenic experience of a T lymphocyte. Mechanisms of homeostasis maintenance of T lymphocytes with different activation phenotypes remain largely unknown. To investigate impact of T cell receptor (TCR) transgenic chains on formation of T lymphocytes, their peripheral survival and activation surface phenotypes, we have generated the transgenic mouse strain expressing transgenic ß-chain of TCR 1D1 (belonging to the Vß6 family) on the genetic background B10.D2(R101). Intrathymic development of T cells in these transgenic mice is not impaired. The repertoire of peripheral T lymphocytes in these mice contains 70-80% of T cells expressing transgenic ß-chain and 20-30% of T cells expressing endogenous ß-chains. The ratio of peripheral CD4⁺CD8⁻ and CD4⁻CD8⁺ T lymphocytes remained unchanged in the transgenic animals, but the percent of T lymphocytes with the "naïve" phenotype CD44⁻CD62L⁺ was significantly increased, whereas the levels of effector memory CD44⁺CD62L⁻ and central memory CD44⁺CD62L⁺ T lymphocytes were markedly decreased in both subpopulations. On the contrary, T lymphocytes expressing endogenous ß-chains had surface phenotype of activated T cells CD44⁺. Thus, for the first time we have shown that the pool of T lymphocytes with different activation phenotypes depends on the structure of T cell receptors.

Production of human lactoferrin in animal milk.

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.

[Generation of transgenic animals, expressing alpha- and beta-chains of autoreactive TCR].

Transgenic animal studies has become a key approach for gene function analysis as well as for modeling of different human diseases, including autoimmune diseases caused by activation of T-lymphocyte clones whose TCRs possesses high affinity for syngeneic MHC molecules. In this study we cloned genes, encoding alpha- and beta- chains of autoreactive TCR of hybridoma 7, specific for syngeneic MHC class II molecules A(b). Amplified DNA fragments, containing rearranged genomic DNA of alpha- and beta-chains of hybridoma 7 were cloned into special cassette vectors, containing natural promoter and enhancer elements for direct expression of genes encoding TCR alpha- and beta-chains in T-lymphocytes of transgenic animals. Using this cassette vectors we generated animals in which most of peripheral T-lymphocytes carry alpha-chain, as well as animals with expression of beta-chain transgene of autoreactive TCR. Obtained animals may serve to explain a number of intrathymic selection processing features and T cell maturation as well as to serve as experimental models for development of new approaches to therapy of autoimmune diseases.

[Analysis of transfer of microinjected zygotes in production of transgenic mice].

The data on transfer of mouse eggs microinjected with DNA during production of transgenic mice were analyzed. The transfer of mouse eggs into both oviducts did not lead to a reliably higher birth rate. It did not affect the frequency of recipients' pregnancy and, although somewhat increased the frequency of multiple birth, led, finally, to unjustified loss of the major part of viable DNA-injected eggs. We recommend transferring no less than 15 microinjected eggs only in one oviduct of each recipient. The transfer into another oviduct is acceptable if the transfer into the first oviduct failed or its outcome is doubtful.

[Transcription and mRNA splicing of the human lactoferrin gene controlled by the regulatory region of the bovine alphaS1 casein gene in the mammary gland of transgenic mice and in mouse embryonic stem cells].

The regulatory region of the bovine alphaS1 casein gene was used to obtain two genetic constructs for expression of human lactoferrin in the mammary gland of transgenic animals. Several transfected mouse embryonic stem cell (ESC) lines and primary transgenic mice were generated with these constructs. Recombinant lactoferrin was not detected in milk of transgenic mice by Western blotting. However, a recombinant transcript was found in RNAs isolated from mammary glands of transgenic females during lactation and from transfected ESC lines.

[A mice model of amyotrophic lateral sclerosis expressing mutant human FUS protein].

UNLABELLED: BACKGROUND AND ОBJECTIVE: Loss of conformation and function of sufficient number of proteins with high aggregation capacity plays an important role in the pathogenesis of many neurodegenerative disorders (NDD). Due to a recent discovery of new array of proteins with the capacity to form aggregates of nonamyloid type, new NDD models as well as a new level of understanding in vivo models which are already exist is needed. DNA/RN A binding proteins - FUS and TDP-43 play a crucial role in the pathogenesis of some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The objective of the study was to develop a new ALS transgenic model. MATERIAL AND METHODS: In cell culture experiments, we studied mutant FUS proteins capable to form intracellular deposits morphologically similar to those observed in the autopsy material of ALS patients. RESULTS AND CONCLUSION: We created a transgenic mice line, in which a pathogenic form of human FUS protein was expressed in the nervous system. That led to the aggregation of FUS protein in spinal cord and motor neurons with the following degeneration and development of a phenotype, similar to the human ALS disease phenotype, in young grown-up animals. This neurodegenerative phenotype corresponds to a great number of clinical manifestations of human ALS and is an adequate transgenic model of the disease.