STAT3 and SMAD1 signaling in Medaka embryonic stem-like cells and blastula embryos.

The activation and transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is essential for maintaining mouse embryonic stem (ES) cell cultures in an undifferentiated state. However, reports from human and monkey ES-cell culture suggest that STAT3 is dispensable for pluripotency in these systems. At the same time, BMP signaling via smad1 was shown to be able to counteract STAT3 signaling in murine ES-cell cultures, while it influences differentiation in multifaceted ways in other cellular contexts. Hence, the question arises whether the signaling situation found in mice or primates and human ES-cells represent the rule or the exception. With this study, we want to contribute an answer to this question from an evolutionary perspective. Therefore, we analyzed the expression and activation status of the Medaka (Oryzias latipes) STAT3 and SMAD1 in Medaka ES-cell-like cultures and their in vivo counterpart, the Medaka blastula embryo. While SMAD signaling is active in the culture system as well as in blastula embryos, our results indicate that STAT3 is inactive and can thus not be involved in pluripotency control of blastula cells or their derived pluripotent in vitro counterparts. These results suggest that the signaling pathways active in the mouse ES-cell culture system represent the exception, while inactivity of STAT3 is apparently the rule in vertebrate ES-cell cultures.

[Discovery of genes active in embryogenesis by gene trapping]. / Obnaruzhenie genov aktivnykh v émbriogeneze posredstvom geneticheskogo tréppinga.

Gene trapping is one of the most promising technologies of vector mutagenesis used for discovery of genes active in embryogenesis. A brief description is provided for gene trapping based on the use of embryonic stem cells and vectors carrying the reporter gene lacZ without promoter, as well as the results obtained with the help of this technology. A total of four transgenic mouse lines were obtained, in three of which the vector was integrated into genes active during embryogenesis. As a result, various patterns of beta-galactosidase expression were observed in the limb rudiments, heart, liver, and other organs at different embryonic stages. At present, detailed studies of the site and time of action of the mutated genes in embryonic and postnatal development are under way, as well as molecular-genetic identification of these genes.

Generation dependent reduction of tTA expression in double transgenic NZL-2/tTA(CMV) mice.

Despite the overall successful application of the tet-system to regulate gene expression in vitro and in vivo, nothing is known so far about the long-term stability of this system in transgenic mice. In this study, mice of generation F2, F3, F4, or F10 of two independent tTA(CMV) transgenic lines were bred with NZL-2 mice containing a tTA-responsive bidirectional promoter that allows the simultaneous expression of two reporter genes encoding luciferase and beta-galactosidase. Analysis of the expression of transgenes in double transgenic mice revealed a dramatic reduction of tTA transactivator mRNA over time. As a consequence, the expression of both reporter genes was gradually reduced from generation to generation in tissues of embryonic and adult NZL-2/tTA(CMV) mice. Luciferase activity in NZL-2/tTA(CMV)(F10) mice was reduced 8-10-fold compared to NZL-2/ tTA(CMV)(F2) mice, and beta-galactosidase expression was no longer detectable. In summary, we describe the long-term instability of the tet-system in our NZL-2/tTA(CMV) double transgenic mice. The molecular basis of this observation and experimental tools to overcome this limitation need to be addressed in future.

Tet-system for the regulation of gene expression during embryonic development.

The ability to control gene expression in a temporal and spatial manner provides a new tool for the study of mammalian gene function particularly during development and oncogenesis. In this study the suitability of the tet-system for investigating embryogenesis was tested in detail. The tTACMV(M1) and rTACMV-3 (reverse Tc-controlled transactivator) transgenic mice were bred with NZL-2 bi-reporter mice containing the vector with a tTA/rTA responsive bidirectional promoter that allows simultaneous regulation of expression of two reporter genes encoding luciferase and beta-galactosidase. In both cases reporter genes were found to be expressed in a wide spectrum of tissues of double transgenic embryos and adult mice. The earliest expression was detected in tTACMV(M1)/NZL-2 embryos at embryonic day 10.5 (E10.5) and rTACMV-3/NZL-2 embryos at E13.5. Doxycycline abolished beta-gal expression in tTACMV(M1)/NZL-2 but induced it in rTACMV-3/NZL-2 embryos including late stages of embryo-genesis. The tTA and rtTA transactivators thus revealed a partially complementary mode of action during second half of embryonic development. These experiments demonstrated that both Tet regulatory systems function during embryonic development. We conclude that the Tet systems allows regulation of gene expression during embryonic development and that 'double reporter' animals like the NZL-2 mice are useful tools for the characterization of newly generated tet transactivator lines expressing tTA (or rtTA) in embryonic as well as in adult tissues.

T lymphocytes of allophenic mice do not reject spleen colony-forming units of the parental genotypes.

Self-tolerance in BALB/c<-->C57BL/6 and BALB/c<-->B10.SM (SM) allophenic mice is examined in this study. Chimerism was determined by coat mosaicism, using electrophoresis of allozyme variants at the glucose phosphate isomerase-1 (GPI-1) in peripheral blood erythrocytes and a cytotoxity test for lymph node (LN) lymphocytes and blood nucleated cells. An age-dependent increase in the percent content of BALB/c erythrocytes was observed in both types of chimeras. The immunological status of chimeras was assessed by T cell interactions with spleen colony-forming cells (SFU-S) able to form colonies in the recipient spleens. LN lymphocytes of the chimeras, together with bone marrow cells (BMC) of the parental genotype, were transplanted to lethally irradiated F1 hybrids. Chimera T lymphocytes did not inactivate CFU-S of the parental genotype and colonies formed in the spleens of F1 hybrids. The absence of splenic colonies in (C x B6)F1 hybrids, in which BMC from B6 had been transplanted (either in combination with chimera lymphocytes or alone), was due to the reaction of host natural killer (NK) cells. The results obtained testify to the permanent immunological tolerance of chimera T lymphocytes to BMC antigens of the both parental genotypes. The chimeric shift in the erythrocyte population is not coupled to disturbances in self-tolerance of chimeric mice.

Lung-targeted expression of the c-Raf-1 kinase in transgenic mice exposes a novel oncogenic character of the wild-type protein.

The c-Raf-1 kinase is a downstream effector of Ras signaling. Both proteins are highly oncogenic when they are mutationally activated, but only the Ras GTPase is frequently mutated in naturally occurring tumors. Although the c-Raf-1 protein was found to be amplified in different lung cancer cell lines, overexpression of the wild-type c-Raf-1 protein was shown to be insufficient to transform cultured cells. Here we have addressed the question of whether overexpression of the wild-type c-Raf-1 kinase can induce lung cancer in mice. We show that lung-targeted expression of oncogenically activated or wild-type c-Raf-1 proteins induces morphologically indistinguishable lung adenomas in transgenic mice. Compared with mice transgenic for the activated c-Raf-1-BxB, tumor development is delayed and occurs at a lower incidence in wild-type c-Raf-1 transgenic mice. Our studies show that the c-Raf-1 expression level is a critical parameter in tumor development and should be analyzed in more detail to evaluate its potential in the induction of cancer.

Measles virus-induced immunosuppression in vitro is associated with deregulation of G1 cell cycle control proteins.

Virus-induced immunosuppression is the major cause of the high morbidity/mortality rates associated with acute measles. It has been shown previously that mitogen-dependent proliferation of peripheral blood lymphocytes (PBL) was strongly impaired after contact with the measles virus (MV) glycoproteins F and H expressed on the surface of infected cells, cells transfected with the corresponding expression constructs or UV-inactivated MV (UV-MV). The state of unresponsiveness was not associated with the induction of apoptosis, and a significant proportion of PBL was found to be arrested in the G0/G1 phase of the cell cycle. It is now shown that cell cycle cessation, rather than complete arrest, is induced after MV glycoprotein contact. No obvious role was found for p53 in the induction of this unresponsiveness. With UV-MV as effector, downregulation of p27, an inhibitor of cyclin-dependent kinase (CDK)-cyclin complexes, was significantly delayed after mitogenic stimulation of human PBL. The activities of both CDK4/6-cyclin D and CDK2-cyclin E complexes for phosphorylation of exogenous substrates in vitro were strongly reduced. CDK4, CDK6, cyclins D3 and E and, to a minor extent, CDK2 failed to accumulate at the protein level after mitogenic stimulation in the presence of UV-MV. These data indicate that MV-induced proliferative unresponsiveness of PBL to mitogenic stimulation is associated with a drastic deregulation of the expression of cell cycle genes essential for the G1/S phase transition.

A comparison of the germline potential of differently aged ES cell lines and their transfected descendants.

The germline transmission (g.l.t.) of gene trap or gene targeted mutations by ES-cell-derived chimaeric mice is a crucial step in the generation of stable transgenic lines. The wild-type ES cell lines CJ7, D3 and R1 of different passage numbers and their transfected clone-descendants generated in gene targeting or gene trap experiments were tested for their ability to colonize the germline. The maximal g.l.t. age for wild-type ES cells was equal to passage 26 and for transfected clones was equivalent to passage 32 of parental lines. It is shown that wild-type ES cells of less than a passage 15 should be used for effective production of transgenic g.l.t. clones. A simple system is outlined to evaluate the probability of g.l.t. on the basis of the chimaeric progeny obtained.

[An analysis of chimeric mice obtained by the injection of the inner cell mass into the blastocyst]. / Analiz khimernykh myshei, poluchennykh putem in''ektsii kletok vnutrennei kletochnoi massy v blastotsistu.

Mouse chimeras were produced using injections of ICM cells into blastocysts. Chimerism of resulting animals was determined by their coat color and spectrum of glucosephosphate isomerase isoenzymes. The use of modifications of the injection method for solving different genetic and embryological problems is discussed.

[Genotypic status of different tissues and organs in aggregated chimeras of BALB/c----C57BL/10 and BALB/c----B10.D2 with discovered chimeric drift]. / Genotipicheskii sostav razlichnykh tkanei i organov u agregatsionnykh khimer myshi BALB/c--C57BL/10 i BALB/c--B10.D2 s vyiavlennym khimernym dreifom.

Genotypic composition of different tissues and organs in BALB/s----C57BL/10 and BALB/c----B10.D2 mice in which chimeric drift was discovered in peripheral blood, erythrocyte population was studied. The proportion of cells of parental components was defined visually in coat, by electrophoresis of allozyme variants at the Gpi-1 locus in blood, spleen, brain, by differential C-staining of X and Y chromosome in cells of bone marrow and by mating of chimeras with BALB/c mice in gametes population. Chimeric drift in the erythrocyte population is not caused by advantage of BALB/c parental component over C57BL parental component in embryonal or postnatal development of whole chimeric organism, but result from selective advantage of hemopoietic BALB/c cells over C57BL cells in chimeric hemopoietic organs.

[Chimeric drift in blood erythrocyte population in BALB/c----C57BL/10 and BALB----B10.D2 mice]. / Khimernyi dreif v populiatsii éritrotsitov krovi u myshei BALB/c----C57Bl/10 i BALB/c----B10.D2.

Genotypic composition of the erythrocyte population of peripheral blood in 16 aggregation chimeras: BALB/c (H-2dd)----C57BL/10(H-2bb) and BALB/c(H-2dd)----B10.D2(H-2dd) was studied during 10 months. The proportion of cells of parental components was defined visually in the coat and by electrophoresis of allozyme variants at the Gpi-1 locus in blood. The similar increase of blood cells percentage was observed in blood of both types of chimeras with age. In chimeras the skin grafts of both parental types survive. Chimeric drift is not caused by H-2 haplotypes differences between cells of two strains or disturbance of immunological tolerance in the chimeric mice. We propose that chimeric drift results from interaction of hemopoietic cells of different strains in early stages of hemopoiesis.