Improved detection of lacZ reporter gene expression in transgenic epithelia by immunofluorescence microscopy

The bacterial lacZ gene is commonly used as a reporter for the in vivo analysis of gene regulation in transgenic mice. However, several laboratories have reported poor detection of beta-galactosidase (the lacZ gene product) using histochemical techniques, particularly in skin. Here we report the difficulties we encountered in assessing lacZ expression in transgenic keratinocytes using classic X-gal histochemical protocols in tissues shown to express the transgene by mRNA in situ hybridization. We found that lacZ reporter gene expression could be reliably detected in frozen tissue sections by immunofluorescence analysis using a beta-galactosidase-specific antibody. Moreover, we were able to localize both transgene and endogenous gene products simultaneously using double-label immunofluorescence. Our results suggest that antibody detection of beta-galactosidase should be used to verify other assays of lacZ expression, particularly where low expression levels are suspected or patchy expression is observed.

Suppressor of cytokine signalling gene expression is elevated in breast carcinoma

Cytokines are important for breast cell function, both as trophic hormones and as mediators of host defense mechanisms against breast cancer. Recently, inducible feedback suppressors of cytokine signalling (SOCS/JAB/SSI) have been identified, which decrease cell sensitivity to cytokines. We examined the expression of SOCS genes in 17 breast carcinomas and 10 breast cancer lines, in comparison with normal tissue and breast lines. We report elevated expression of SOCS-1-3 and CIS immunoreactive proteins within in situ ductal carcinomas and infiltrating ductal carcinomas relative to normal breast tissue. Significantly increased expression of SOCS-1-3 and CIS transcripts was also shown by quantitative in situ hybridisation within both tumour tissue and reactive stroma. CIS transcript expression was elevated in all 10 cancer lines, but not in control lines. However, there was no consistent elevation of other SOCS transcripts. CIS protein was shown by immunoblot to be present in all cancer lines at increased levels, mainly as the 47 kDa ubiquitinylated form. A potential proliferative role for CIS overexpression is supported by reports that CIS activates ERK kinases, and by strong induction in transient reporter assays with an ERK-responsive promoter. The in vivo elevation of SOCS gene expression may be part of the host/tumour response or a response to autocrine/paracrine GH and prolactin. However, increased CIS expression in breast cancer lines appears to be a specific lesion, and could simultaneously shut down STAT 5 signalling by trophic hormones, confer resistance to host cytokines and increase proliferation through ERK kinases.

Transcriptional regulation of CD4 gene expression by T cell factor-1/beta-catenin pathway.

By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/beta-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/beta-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+ CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1(-/-) DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1(-/-) cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with beta-catenin. Furthermore, forced expression of a stabilized beta-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized beta-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and beta-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/beta-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.

Aggregate cultures of foetal rat liver cells: development and maintenance of liver gene expression.

Rotation-mediated aggregate cultures of foetal rat liver cells were prepared and grown in a chemically defined medium. Their capacity for cellular organisation and maturation was studied over a culture period of 3 wk by using both morphologic and biochemical criteria. It was found that within each aggregate, distinct liver cell types were present and attained their normal, differentiated phenotype. Parenchymal cells formed small acini with a central lumen. Within the first 2 wk in culture, albumin and ferritin mRNA levels were maintained, while the alpha-fetoprotein mRNA levels decreased, and tyrosine aminotransferase (TAT) gene expression increased. No significant response to glucocorticoids was observed in early cultures, whereas after 3 wk a marked increase in TAT mRNA levels was elicited by dexamethasone and glucagon (additive stimulatory effects). The results show that foetal rat liver cells cultured in a chemically defined medium are able to rearrange themselves into histotypic structures, and display a developmental pattern of gene expression comparable to that of perinatal rat liver in vivo. This culture system offers therefore a useful model to study the development and function of liver cells.

Estrogen receptor regulates MyoD gene expression by preventing AP-1-mediated repression.

Cell growth and differentiation are opposite events in the myogenic lineage. Growth factors block the muscle differentiation program by inducing the expression of transcription factors that negatively regulate the expression of muscle regulatory genes like MyoD. In contrast, extracellular clues that induce cell cycle arrest promote MyoD expression and muscle differentiation. Thus, the regulation of MyoD expression is critical for muscle differentiation. Here we show that estrogen induces MyoD expression in mouse skeletal muscle in vivo and in dividing myoblasts in vitro by relieving the MyoD promoter from AP-1 negative regulation through a mechanism involving estrogen receptor/AP-1 protein-protein interactions but independent of the estrogen receptor DNA binding activity.

Selective and powerful stress gene expression in Arabidopsis in response to malondialdehyde.

The provenance, half-life and biological activity of malondialdehyde (MDA) were investigated in Arabidopsis thaliana. We provide genetic confirmation of the hypothesis that MDA originates from fatty acids containing more than two methylene-linked double bonds, showing that tri-unsaturated fatty acids are the in vivo source of up to 75% of MDA. The abundance of the combined pool of free and reversibly bound MDA did not change dramatically in stress, although a significant increase in the free MDA pool under oxidative conditions was observed. The half-life of infiltrated MDA indicated rapid metabolic turnover/sequestration. Exposure of plants to low levels of MDA using a recently developed protocol powerfully upregulated many genes on a cDNA microarray with a bias towards those implicated in abiotic/environmental stress (e.g. ROF1 and XERO2). Remarkably, and in contrast to the activities of other reactive electrophile species (i.e. small vinyl ketones), none of the pathogenesis-related (PR) genes tested responded to MDA. The use of structural mimics of MDA isomers suggested that the propensity of the molecule to act as a cross-linking/modifying reagent might contribute to the activation of gene expression. Changes in the concentration/localisation of unbound MDA in vivo could strongly affect stress-related transcription.

Inducible gene expression in fetal thymic epithelium: A new BAC transgenic model.

The thymus is the site of T cell development. Several stromal and hematopoietic cell types are necessary for the proper function of thymic selection and eventually peripheral immunity. Thymic epithelial cells (TECs) are essential for T cell lineage commitment, expansion, and maturation in the thymus. We were interested in developing an in vivo model in which exogenous gene expression could be transiently induced in embryonic TEC (Tet-On system). To this end, we have generated a bacterial artificial chromosome (BAC) transgenic mouse line in which the reverse tetracycline-dependent transactivator (rtTA) is expressed under the control of the Foxn1 promoter, a transcriptional factor indispensable for TEC development. To analyze the expression pattern and efficiency of this novel mouse model, we crossed the Foxn1-rtTA founder with a Tet-Responsive Element (TRE)-LacZ GFP mouse reporter to obtain a double transgenic mouse. In the presence of doxycycline, rtTA can interact with TRE and induce the expression of GFP and LacZ. In this double transgenic mouse, we observed that GFP expression was high, inducible and limited to TEC in fetal thymus. In contrast, in adult thymus, when TEC development and maturation is completed, GFP was barely detectable. Therefore, Foxn1-rtTA represents a new and efficient transgenic mouse model to induce genes of interest specifically in fetal thymic epithelium. genesis 51:717-724. © 2013 Wiley Periodicals, Inc.

Real-time recording of circadian liver gene expression in freely moving mice reveals the phase-setting behavior of hepatocyte clocks.

The mammalian circadian timing system consists of a master pacemaker in the suprachiasmatic nucleus (SCN) in the hypothalamus, which is thought to set the phase of slave oscillators in virtually all body cells. However, due to the lack of appropriate in vivo recording technologies, it has been difficult to study how the SCN synchronizes oscillators in peripheral tissues. Here we describe the real-time recording of bioluminescence emitted by hepatocytes expressing circadian luciferase reporter genes in freely moving mice. The technology employs a device dubbed RT-Biolumicorder, which consists of a cylindrical cage with reflecting conical walls that channel photons toward a photomultiplier tube. The monitoring of circadian liver gene expression revealed that hepatocyte oscillators of SCN-lesioned mice synchronized more rapidly to feeding cycles than hepatocyte clocks of intact mice. Hence, the SCN uses signaling pathways that counteract those of feeding rhythms when their phase is in conflict with its own phase.

Two waves of recombinase gene expression in developing thymocytes.

During T cell development in the thymus, T cell receptor (TCR) alpha, beta, gamma, and delta genes are rearranged and expressed. TCR rearrangement strictly depends upon the coordinate activity of two recombinase activating genes, Rag-1 and Rag-2. In this study we have followed the expression of these genes at different stages of intrathymic development. The results indicate that there are two periods of high Rag-1 and Rag-2 mRNA expression. The first wave peaks early at the CD25+CD4-CD8-CD3- stage of development and coincides with the initial appearance of transcripts derived from fully rearranged TCR beta, gamma, and delta genes, whereas the second wave occurs later at the CD4+CD8+ stage coincident with full-length TCR alpha mRNA expression. Active downregulation of Rag-1 and Rag-2 mRNA expression appears to occur in vivo between the two peaks of recombinase activity. This phenomenon can be mimicked in vitro in response to artificial stimuli such as phorbol myristate acetate and calcium ionophore. Collectively our data suggest that recombinase expression is actively regulated during early thymus development independently of cell surface expression of a mature heterodimeric TCR protein complex.

Long-term cerebral plasticity-related gene expression : a study of the respective role of CBP and CRTC1 in CREB transcriptional activation

1.1 AbstractThe treatment of memory disorders and cognitive deficits in various forms of mental retardation may greatly benefit from a better understanding of the molecular and cellular mechanisms of memory formation. Different forms of memory have distinct molecular requirements.Short-term memory (STM) is thought to be mediated by covalent modifications of existing synaptic molecules, such as phosphorylation or dephosphorylation of enzymes, receptors or ion channels. In contrast, long-term memoiy (LTM) is thought to be mediated by growth of new synapses and restructuring of existing synapses. There is extensive evidence that changes in gene expression and de novo protein synthesis are key processes for LTM formation. In this context, the transcription factor CREB (cAMP-response element-binding protein) was shown to be crucial. Activation of CREB requires phosphorylation of a serine residue (Ser-133), and the subsequent recruitment of a coactivator called CREB-binding protein (CBP). Moreover, we have recently shown that another coactivator called CREB Regulated Transcription Coactivator 1 (CRTC1) functions as a calcium- and cAMP-sensitive coincidence detector in neurons, and is involved in hippocampal long-term synaptic plasticity. Given the importance of cAMP and calcium signaling for plasticity-related gene expression in neurons and in astrocytes, we sought to determine the respective involvement of the CREB coactivators CBP and CRTC1 in CREB-mediated transcription.We developed various strategies to selectively interfere with these CREB coactivators in mouse primary neurons and in astrocytes in vitro. However, despite several pieces of evidence implicating CBP and/or CRTC1 in the regulation of neuronal plasticity genes, we could not clearly determine the respective requirement of these coactivators for the activation of these genes. Nevertheless, we showed that calcineurin activity, which is important for CRTC1 nuclear translocation, is necessary for the expression of some CREB-regulated plasticity genes. We associated this phenomena to physiopathological conditions observed in Down's syndrome. In addition, we demonstrated that in astrocytes, noradrenaline stimulates CREB-target gene expression through β-adrenergic receptor activation, intracellular cAMP pathway activation, and CRTC-induced CREB transactivation.Defining the respective role of CREB and its coactivators CBP and CRTC1 in neuronal and astrocytic cultures in vitro sets the stage for future in vivo studies and for the possible development of new therapeutic strategies to improve the treatment of memoiy and cognitive disorders.1.2 RésuméUne meilleure connaissance des mécanismes moléculaires et cellulaires responsables de la formation de la mémoire pourrait grandement améliorer le traitement des troubles de la mémoire ainsi que des déficits cognitifs observés dans différentes formes de pathologies psychiatriques telles que le retard mental. Les différentes formes de mémoire dépendent de processus moléculaires différents.La mémoire à court terme (STM) semble prendre forme suite à des modifications covalentes de molécules synaptiques préexistantes, telles que la phosphorylation ou la déphosphorylation d'enzymes, de récepteurs ou de canaux ioniques. En revanche, la mémoire à long terme (LTM) semble être due à la génération de nouvelles synapses et à la restructuration des synapses existantes. De nombreuses études ont permis de démontrer que les changements dans l'expression des gènes et la synthèse de protéine de novo sont des processus clés pour la formation de la LTM. Dans ce contexte, le facteur de transcription CREB (cAMP-response element-binding protein) s'est avéré être un élément crucial. L'activation de CREB nécessite la phosphorylation d'un résidu sérine (Ser-133), et le recrutement d'un coactivateur nommé CBP (CREB binding protein). En outre, nous avons récemment démontré qu'un autre coactivateur de CREB nommé CRTC1 (CREB Regulated Transcription Coactivator 1) agit comme un détecteur de coïncidence de l'AMP cyclique (AMPc) et du calcium dans les neurones et qu'il est impliqué dans la formation de la plasticité synaptique à long terme dans l'hippocampe. Etant donné l'importance des voies de l'AMPc et du calcium dans l'expression des gènes impliqués dans la plasticité cérébrale, nous voulions déterminer le rôle respectif des coactivateurs de CREB, CBP et CRTC1.Nous avons développé diverses stratégies pour interférer de façon sélective avec les coactivateurs de CREB dans les neurones et dans les astrocytes chez la souris in vitro. Nos résultats indiquent que CBP et CRTC1 sont tous deux impliqués dans la transcription dépendante de CREB induite par l'AMPc et le calcium dans les neurones. Cependant, malgré plusieurs évidences impliquant CBP et/ou CRTC1 dans l'expression de gènes de plasticité neuronale, nous n'avons pas pu déterminer clairement leur nécessité respective pour l'activation de ces gènes. Toutefois, nous avons montré que l'activité de la calcineurine, dont dépend la translocation nucléaire de CRTC1, est nécessaire à l'expression de certains de ces gènes. Nous avons pu associer ce phénomène à une condition physiopathologique observée dans le syndrome de Down. Nous avons également montré que dans les astrocytes, la noradrénaline stimule l'expression de gènes cibles de CREB par une activation des récepteurs β- adrénergiques, l'activation de la voie de l'AMPc et la transactivation de CREB par les CRTCs.Définir le rôle respectif de CREB et de ses coactivateurs CBP et CRTC1 dans les neurones et dans les astrocytes in vitro permettra d'acquérir les connaissances nécessaires à de futures études in vivo et, à plus long terme d'éventuellement développer des stratégies thérapeutiques pour améliorer les traitements des troubles cognitifs.

Reduced phototropism in pks mutants may be due to altered auxin-regulated gene expression or reduced lateral auxin transport.

Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1- and phot2-mediated phototropism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi-reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution.

Requirement of glucose metabolism for regulation of glucose transporter type 2 (GLUT2) gene expression in liver.

Previous studies have shown that glucose increases the glucose transporter (GLUT2) mRNA expression in the liver in vivo and in vitro. Here we report an analysis of the effects of glucose metabolism on GLUT2 gene expression. GLUT2 mRNA accumulation by glucose was not due to stabilization of its transcript but rather was a direct effect on gene transcription. A proximal fragment of the 5' regulatory region of the mouse GLUT2 gene linked to a reporter gene was transiently transfected into liver GLUT2-expressing cells. Glucose stimulated reporter gene expression in these cells, suggesting that glucose-responsive elements were included within the proximal region of the promoter. A dose-dependent effect of glucose on GLUT2 expression was observed over 10 mM glucose irrespective of the hexokinase isozyme (glucokinase K(m) 16 mM; hexokinase I K(m) 0.01 mM) present in the cell type used. This suggests that the correlation between extracellular glucose and GLUT2 mRNA concentrations is simply a reflection of an activation of glucose metabolism. The mediators and the mechanism responsible for this response remain to be determined. In conclusion, glucose metabolism is required for the proper induction of the GLUT2 gene in the liver and this effect is transcriptionally regulated.

Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1.

The ability to regulate specific genes of energy metabolism in response to fasting and feeding is an important adaptation allowing survival of intermittent food supplies. However, little is known about transcription factors involved in such responses in higher organisms. We show here that gene expression in adipose tissue for adipocyte determination differentiation dependent factor (ADD) 1/sterol regulatory element binding protein (SREBP) 1, a basic-helix-loop-helix protein that has a dual DNA-binding specificity, is reduced dramatically upon fasting and elevated upon refeeding; this parallels closely the regulation of two adipose cell genes that are crucial in energy homeostasis, fatty acid synthetase (FAS) and leptin. This elevation of ADD1/SREBP1, leptin, and FAS that is induced by feeding in vivo is mimicked by exposure of cultured adipocytes to insulin, the classic hormone of the fed state. We also show that the promoters for both leptin and FAS are transactivated by ADD1/SREBP1. A mutation in the basic domain of ADD1/SREBP1 that allows E-box binding but destroys sterol regulatory element-1 binding prevents leptin gene transactivation but has no effect on the increase in FAS promoter function. Molecular dissection of the FAS promoter shows that most if not all of this action of ADD1/SREBP1 is through an E-box motif at -64 to -59, contained with a sequence identified previously as the major insulin response element of this gene. These results indicate that ADD1/SREBP1 is a key transcription factor linking changes in nutritional status and insulin levels to the expression of certain genes that regulate systemic energy metabolism.

Critical role for Ets, AP-1 and GATA-like transcription factors in regulating mouse Toll-like receptor 4 (Tlr4) gene expression.

TLR4 (Toll-like receptor 4) is essential for sensing the endotoxin of Gram-negative bacteria. Mutations or deletion of the TLR4 gene in humans or mice have been associated with altered predisposition to or outcome of Gram-negative sepsis. In the present work, we studied the expression and regulation of the Tlr4 gene of mouse. In vivo, TLR4 levels were higher in macrophages compared with B, T or natural killer cells. High basal TLR4 promoter activity was observed in RAW 264.7, J774 and P388D1 macrophages transfected with a TLR4 promoter reporter vector. Analysis of truncated and mutated promoter constructs identified several positive [two Ets (E twenty-six) and one AP-1 (activator protein-1) sites] and negative (a GATA-like site and an octamer site) regulatory elements within 350 bp upstream of the transcriptional start site. The myeloid and B-cell-specific transcription factor PU.1 bound to the proximal Ets site. In contrast, none among PU.1, Ets-1, Ets-2 and Elk-1, but possibly one member of the ESE (epithelium-specific Ets) subfamily of Ets transcription factors, bound to the distal Ets site, which was indispensable for Tlr4 gene transcription. Endotoxin did not affect macrophage TLR4 promoter activity, but it decreased TLR4 steady-state mRNA levels by increasing the turnover of TLR4 transcripts. TLR4 expression was modestly altered by other pro- and anti-inflammatory stimuli, except for PMA plus ionomycin which strongly increased promoter activity and TLR4 mRNA levels. The mouse and human TLR4 genes were highly conserved. Yet, notable differences exist with respect to the elements implicated in gene regulation, which may account for species differences in terms of tissue expression and modulation by microbial and inflammatory stimuli.

MAR-mediated integration of plasmid vectors for in vivo gene transfer and regulation.

BACKGROUND: The in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models. RESULTS: In this study, we devised vectors allowing a tight regulation of transgene expression in mice from such non-viral vectors using a doxycycline-controlled network of activator and repressor proteins. Using these vectors, we demonstrate proper physiological response as consequence of the induced expression of two therapeutically relevant proteins, namely erythropoietin and utrophin. Kinetic studies showed that the induction of transgene expression was only transient, unless epigenetic regulatory elements termed Matrix Attachment Regions, or MAR, were inserted upstream of the regulated promoters. Using episomal plasmid rescue and quantitative PCR assays, we observed that similar amounts of plasmids remained in muscles after electrotransfer with or without MAR elements, but that a significant portion had integrated into the muscle fiber chromosomes. Interestingly, the MAR elements were found to promote plasmid genomic integration but to oppose silencing effects in vivo, thereby mediating long-term expression. CONCLUSIONS: This study thus elucidates some of the determinants of transient or sustained expression from the use of non-viral regulated vectors in vivo.