Biomedical subjects

S Pournin

Publications and source records attributed to S Pournin.

15 recordsLinked to original sources

PubMed1996

Cardiovascular lesions and skeletal myopathy in mice lacking desmin.

In order to further our understanding of the biological role of desmin, the muscle-specific intermediate filament protein, a null mutation in the desmin gene was introduced into the germ line of mice. Despite the complete lack of desmin, these mice developed and reproduced. Since we show that skeletal, cardiac, and smooth muscles form in the Des-/- mice, it is reasonable to propose that desmin is not essential for myogenic commitment or for myoblast fusion or differentiation in vivo. However, morphological abnormalities were observed in the diaphragm of adult mice; these were demonstrated by disorganized, distended, and nonaligned fibers. The heart presented areas of hemorrhaging in which fibrosis and ischemia were observed. We have also shown that the absence of desmin produces specific defects in smooth muscles. In conclusion, our results have demonstrated that desmin is not required for the differentiation of skeletal, cardiac, and smooth muscles but is essential to strengthen and maintain the integrity of these tissues.

Animals↗

PubMed1996

Gene targeting the myf-5 locus with nlacZ reveals expression of this myogenic factor in mature skeletal muscle fibres as well as early embryonic muscle.

We have introduced the nlacZ reporter gene into the locus of the myogenic factor gene myf-5 by homologous recombination in embryonic stem (ES) cells. Targeted ES clones were injected into precompaction morula, and the beta-galactosidase expression pattern was monitored. These mice permit the sensitive visualization of myf-5 expression throughout the embryo, and provide a standard for comparing it with that seen with different myf-5/nlacZ transgenes. Thus, in a comparison using ES cells in chimaeric embryos containing the targeted or randomly integrated myf-5/nlacZ construct, we demonstrate that 5.5 kbp of myf-5 upstream flanking sequence including exon1 and most of intron1 directs some skeletal muscle expression, but this is neither qualitatively nor quantitatively equivalent to that of the endogenous gene. Myf-5 is expressed early, before terminal myogenesis takes place in the medial half of the somite, and subsequently it is a major myogenic factor as skeletal muscle forms. All skeletal muscle shows beta-galactosidase activity, even after birth, indicating that myf-5 expression is not confined to primary myotubes, which are derived from embryonic myoblasts, but is also present in muscles containing different adult fibre types. The presence of myf-5 transcripts from the endogenous gene in older muscle was confirmed by in situ hybridization. These results suggest that the myf-5 gene is not activated in only a subset of muscle cells and are consistent with the results on the MyoD knockout mice.

Animals↗

PubMed1996

A high-resolution map around the locus Om on mouse Chromosome 11.

The locus Om (ovum mutant) identified in the mouse strain DDK affects the viability of (DDK x non-DDK)F1 preimplantation embryos. We previously located this locus on Chromosome (Chr) 11 close to Scya2 (Baldacci et al. Mamm. Genome 2, 100-105, 1992). Here we report a high-resolution map of the region around Om based on a large number of backcross individuals. The same region has been analyzed on the EUCIB backcross, and the two maps have been compared. The results define the proximal and distal boundaries for the Om mutation as Scya2 and D11Mit36 respectively. The distance between these two markers is about 2 cM. These data should facilitate the positional cloning and molecular characterization of Om.

Animals↗

PubMed1995

Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice lacking MAOA.

Deficiency in monoamine oxidase A (MAOA), an enzyme that degrades serotonin and norepinephrine, has recently been shown to be associated with aggressive behavior in men of a Dutch family. A line of transgenic mice was isolated in which transgene integration caused a deletion in the gene encoding MAOA, providing an animal model of MAOA deficiency. In pup brains, serotonin concentrations were increased up to ninefold, and serotonin-like immunoreactivity was present in catecholaminergic neurons. In pup and adult brains, norepinephrine concentrations were increased up to twofold, and cytoarchitectural changes were observed in the somatosensory cortex. Pup behavioral alterations, including trembling, difficulty in righting, and fearfulness were reversed by the serotonin synthesis inhibitor parachlorophenylalanine. Adults manifested a distinct behavioral syndrome, including enhanced aggression in males.

Aggression↗

PubMed1995

Developmental expression of H-2K major histocompatibility complex class I transgenes requires the presence of proximal introns.

The pattern of expression of the H-2K major histocompatibility complex (MHC) class I gene is complex. During embryonic development H-2K mRNA, detectable from midgestation, is poorly expressed. In the adult, H-2K expression is nearly ubiquitous but transcriptional regulation occurs leading to different mRNA levels in the different organs of the mouse. In vitro studies have shown that most of the regulatory elements controlling H-2K gene transcription are located in the 5' region of the gene. However, using fusion transgenes in which reporter genes were under the control of 2 kb of H-2K 5' regulatory region, we have previously shown that this region was not sufficient to ensure correct developmental transgene expression. By contrast, a native 9.25 kb H-2K transgene was expressed appropriately both in the adult and in the embryo. In order to localise more precisely the cis-acting regulatory sequences involved in H-2K developmental expression, we have now constructed new transgenic lines containing H-2Kb transgenes that were deleted from specific parts of the H-2Kb gene. We show that deletion of either the H-2K 3' flanking region or the 5 (out of 7) distal introns results in an expression of the transgenes which is similar to that of the endogenous H-2K gene, both in the adult and during embryonic development. By contrast, deletion of all the introns or of the two proximal ones abrogates H-2K transgene expression. Our data reveal the complexity of H-2K regulation and highlight the crucial role of proximal introns in H-2K expression in the living organism.

Animals↗

PubMed1995

Both coding exons of the c-myc gene contribute to its posttranscriptional regulation in the quiescent liver and regenerating liver and after protein synthesis inhibition.

In vivo, the steady-state level of c-myc mRNA is mainly controlled by posttranscriptional mechanisms. Using a panel of transgenic mice in which various versions of the human c-myc proto-oncogene were under the control of major histocompatibility complex H-2Kb class I regulatory sequences, we have shown that the 5' and the 3' noncoding sequences are dispensable for obtaining a regulated expression of the transgene in adult quiescent tissues, at the start of liver regeneration, and after inhibition of protein synthesis. These results indicated that the coding sequences were sufficient to ensure a regulated c-myc expression. In the present study, we have pursued this analysis with transgenes containing one or the other of the two c-myc coding exons either alone or in association with the c-myc 3' untranslated region. We demonstrate that each of the exons contains determinants which control c-myc mRNA expression. Moreover, we show that in the liver, c-myc exon 2 sequences are able to down-regulate an otherwise stable H-2K mRNA when embedded within it and to induce its transient accumulation after cycloheximide treatment and soon after liver ablation. Finally, the use of transgenes with different coding capacities has allowed us to postulate that the primary mRNA sequence itself and not c-Myc peptides is an important component of c-myc posttranscriptional regulation.

Animals↗

PubMed1994

Mice lacking vimentin develop and reproduce without an obvious phenotype.

To address the biological role of vimentin in the context of the living organism, we have introduced a null mutation of the vimentin gene into the germ line of mice. Surprisingly, animals homozygous for this mutation developed and reproduced without an obvious phenotype. Immunoblotting, immunofluorescence, and immunogold labeling analysis confirmed the absence of vimentin and of the corresponding filament network. Furthermore, no compensatory expression of another intermediate filament could be demonstrated. While these results leave open the question of the possible role of vimentin in unusual situations or pathological conditions, they show that a conspicuous developmental and cell-specific structure that is an integral part of the cytoskeleton can be eliminated without apparent effect on mouse reproduction and development.

Animals↗

PubMed1994

Normal and pathological expression of GFAP promoter elements in transgenic mice.

The expression of the glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. In order to characterize DNA sequences involved in such regulations, we produced transgenic mice bearing 2 kb of the 5' flanking region of the murine GFAP gene linked to the Escherichia coli beta-galactosidase (beta-gal) reporter gene. Seven transgenic lines were obtained. We observed that the regulatory elements present in the transgene GFAP-nls-LacZ direct an expression in the neural and non-neural tissue and target in vivo an unexpected subpopulation of astrocyte. In the developing brain, beta-gal activity and GFAP appeared simultaneously and in the same region, on embryonic day 18 (E18), suggesting that the 2 kb of the promoter contains the regulatory sequences responsible for the perinatal vimentin/GFAP switch. In addition, we demonstrated that the 2 kb sequence of the GFAP promoter used in the transgene possess elements which are activated after a surgical injury, thus permitting to study some aspects of reactive gliosis in these transgenic mice. These transgenic lines provide a useful tool by enabling further studies of astroglial and, probably, neuronal physiologies.

Animals↗

PubMed1994

Immortalization of multiple cell types from transgenic mice using a transgene containing the vimentin promoter and a conditional oncogene.

Differentiated clonal cell lines were obtained from transgenic mice carrying a recombinant gene composed of DNA coding for a temperature-sensitive mutant of the simian virus large T antigen under the control of regulatory elements of the human vimentin gene. In response to mitogenic factors the vimentin promoter is activated in the presence of serum in almost all cultured cells independently of their origin. The expression of the T antigen could be controlled both at the level of transcription since the vimentin promoter is growth-regulated and at the level of the protein structure through the temperature stability of the T antigen. Indeed, the switch-off of the oncogene protein is obtained by serum deprivation of the culture and achieved with enhancement of the growth temperature. From transgenic mice several types of clonal differentiated cell lines were established and characterized including melanocytes, macrophages, mesangial, muscle, and endothelial cells. Melanocytes displayed melanin while endothelial cells from brain and heart expressed the related factor VIII and low density lipoprotein absorption capacities. Mesangial cells from kidney exhibited numerous desmosomes. Typical markers of macrophages from bone marrow were observed while skeletal muscle cells fused and contracted.

Animals↗

PubMed1994

A maternal factor affecting mouse blastocyst formation.

Normal development of the mouse embryo requires the presence of both paternal and maternal genomes. This is due to functional differences having their origin in a differential imprinting of parental genomes. Furthermore, several lines of evidence show that the very early interactions between egg cytoplasm and pronuclei may influence the programming of the embryonic genome and modulate the functional inequality of the parental contribution even during preimplantation stages. In this paper, we show that a factor present in ovulated oocytes of the mouse mutant strain DDK and therefore of maternal origin prevents the formation of the blastocyst. This factor, which acts via an interaction with the paternal genome, is present in oocytes as an RNA and is still active in preimplantation embryos. This is the first direct evidence of such a maternal control in the mouse.

Animals↗

PubMed1994

The cis-acting elements known to regulate c-myc expression ex vivo are not sufficient for correct transcription in vivo.

Much of our knowledge about the regulation of the c-myc proto-oncogene expression has come from studies of c-myc gene expression in several well defined ex vivo systems, including differentiation systems and tumor cells. However, very few investigations have been performed to determine the factors and cis-acting sequences that regulate c-myc expression in vivo. In order to obtain information on the sequences required to regulate c-myc gene transcription from the two major P1 and P2 initiation sites in the mouse, we have generated several constructs containing human or murine c-myc genomic sequences with various 5' flanking sequences and derived corresponding transgenic mice. A sensitive S1 nuclease protection assay was performed to analyse and to compare transgene expression with that of the endogenous c-myc mRNA, either in adult organs, or during development. None of the transgenic mice expressed the construct appropriately, although several strains exhibited unexpected expression most probably due to position effects. Our results indicate that the cis-acting elements described to regulate c-myc expression ex vivo are not sufficient to drive the correct expression of c-myc gene in vivo and strongly suggest that additional regulatory elements located upstream from -3500 (with respect to mouse P1 promoter) and downstream 1500 bp from polyadenylation sites are required.

Animals↗

PubMed1993

Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain.

The zinc finger gene Krox-20 is transcribed in two alternate segments (rhombomeres) of the developing hindbrain. To investigate its function, we have used homologous recombination to generate mice carrying an in-frame insertion of the E. coli lacZ gene within Krox-20. Analysis of the beta-galactosidase pattern in heterozygous embryos confirmed the known profile with expression restricted to rhombomeres (r) 3 and 5. Mice homozygous for the mutation die during the first two weeks after birth. Anatomical analysis of the hindbrain and of the cranial nerves during embryogenesis, combined with the determination of the expression patterns of rhombomere-specific genes, demonstrated that Krox-20 inactivation results in a marked reduction or elimination of r3 and r5. We conclude that Krox-20, although not required for the initial delimitation of r3 and r5, plays an important role in the process of segmentation governing hindbrain development.

Aging↗

PubMed1993

The proximal region of the MBP gene promoter is sufficient to induce oligodendroglial-specific expression in transgenic mice.

To characterize regulatory DNA sequences involved in oligodendroglial expression of myelin basic protein (MBP), transgenic mice carrying a 256 bp fragment of the mouse MBP promoter fused to an Escherichia coli lacZ gene were generated. Of four transgenic families, two (lines 2 and 4) expressed beta-galactosidase activity in the nervous system but not in most other tissues. Histochemical and immunohistochemical analysis of adult brain from these two lines showed oligodendroglial-specific expression of the transgene. In line 2, only a small proportion of oligodendrocytes expressed the transgene, and in labelled cells the product of the enzymatic reaction with beta-galactosidase was confined to a small round vesicle in the vicinity of the nucleus. In contrast, in tissue sections from line 4 adult brain and spinal cord beta-galactosidase activity was much more intense and at least 80-90% of oligodendrocytes expressed the transgene. Detection of the MBP-lacZ transcript by in situ hybridization showed that the transgene mRNA was confined to the oligodendrocyte cell body. These results suggest that cis-acting regulatory elements, specifying oligodendrocytes identity, are located within 256 bp upstream from the MBP gene.

Aging↗

PubMed1993

The intronic immunoglobulin kappa gene enhancer acts independently on rearrangement and on transcription.

It is not known which cis-acting elements regulate kappa gene rearrangement. However, the onset of rearrangement coincides with the onset of transcription suggesting that common regulatory elements are used. We have investigated the role of the intronic enhancer on rearrangement using mice transgenic for kappa minigenes. The rabbit kappa enhancer, which is defective in activation of transcription in mouse cells, allows a high level of rearrangement. However, transgenes which possess no enhancer region at all are rearranged a hundred times less compared to transgenes which possess the rabbit enhancer. Our results suggest that while the enhancer region can activate rearrangement as well as transcription, its action on both phenomena can be uncoupled.

Animals↗

PubMed1993

The 5' and 3' non-coding sequences of the c-myc gene, required in vitro for its post-transcriptional regulation, are dispensable in vivo.

We have previously shown that in vivo the steady-state level of c-myc mRNA in different quiescent organs and its induction in the early stages of hepatic regeneration and after inhibition of protein synthesis are mainly controlled by post-transcriptional mechanisms. In order to localize the target sequences for these mechanisms, transgenic lines expressing various versions of the human c-myc proto-oncogene have been constructed. To avoid all possible transcriptional controls due to the c-myc 5' regulatory region, the c-myc genomic sequences were fused to MHC H-2Kb class I regulatory sequences, which have previously been shown to be able to drive reporter gene expression in most adult tissues. The transgenes contained either all human c-myc genomic sequences or were deleted for one of the sequences which have been shown in in vitro experiments to play a role in c-myc mRNA stabilization, in particular exon 1, intron 1 and the 3' non-coding region. Several independent transgenic lines were derived for each construct. Using S1 nuclease protection analysis, we have monitored H-2K, mouse c-myc and transgene mRNA expression in several quiescent adult organs, at the start of liver regeneration and after inhibition of protein synthesis in each transgenic line. Our results indicate that the 5' non-coding sequences, including exon 1 and intron 1, and the 3' untranslated region are all dispensable in the different aspects of c-myc post-transcriptional regulation.

Animals↗