Low-grade and high-grade mammary carcinomas in WAP-T transgenic mice are independent entities distinguished by Met expression.

Mammary carcinomas developing in SV40 transgenic WAP-T mice arise in two distinct histological phenotypes: as differentiated low-grade and undifferentiated high-grade tumors. We integrated different types of information such as histological grading, analysis of aCGH-based gene copy number and gene expression profiling to provide a comprehensive molecular description of mammary tumors in WAP-T mice. Applying a novel procedure for the correlation of gene copy number with gene expression on a global scale, we observed in tumor samples a global coherence between genotype and transcription. This coherence can be interpreted as a matched transcriptional regulation inherited from the cells of tumor origin and determined by the activity of cancer driver genes. Despite common recurrent genomic aberrations, e.g. gain of chr. 15 in most WAP-T tumors, loss of chr. 19 frequently occurs only in low-grade tumors. These tumors show features of "basal-like" epithelial differentiation, particularly expression of keratin 14. The high-grade tumors are clearly separated from the low-grade tumors by strong expression of the Met gene and by coexpression of epithelial (e.g. keratin 18) and mesenchymal (e.g. vimentin) markers. In high-grade tumors, the expression of the nonmutated Met protein is associated with Met-locus amplification and Met activity. The role of Met as a cancer driver gene is supported by the contribution of active Met signaling to motility and growth of mammary tumor-derived cells. Finally, we discuss the independent origin of low- and high-grade tumors from distinct cells of tumor origin, possibly luminal progenitors, distinguished by Met gene expression and Met signaling.

Age-dependent impairment of spine morphology and synaptic plasticity in hippocampal CA1 neurons of a presenilin 1 transgenic mouse model of Alzheimer's disease.

Presenilin 1 (PS1) mutations are responsible for a majority of early onset familial Alzheimer's disease (FAD) cases, in part by increasing the production of Abeta peptides. However, emerging evidence suggests other possible effects of PS1 on synaptic dysfunction where PS1 might contribute to the pathology independent of Abeta. We chose to study the L286V mutation, an aggressive FAD mutation which has never been analyzed at the electrophysiological and morphological levels. In addition, we analyzed for the first time the long term effects of wild-type human PS1 overexpression. We investigated the consequences of the overexpression of either wild-type human PS1 (hPS1) or the L286V mutated PS1 variant (mutPS1) on synaptic functions by analyzing synaptic plasticity and associated spine density changes from 3 to 15 months of age. We found that mutPS1 induces a transient increase observed only in 4- to 5-month-old mutPS1 animals in NMDA receptor (NMDA-R)-mediated responses and LTP compared with hPS1 mice and nontransgenic littermates. The increase in synaptic functions is concomitant with an increase in spine density. With increasing age, however, we found that the overexpression of human wild-type PS1 progressively decreased NMDA-R-mediated synaptic transmission and LTP, without neurodegeneration. These results identify for the first time a transient increase in synaptic function associated with L286V mutated PS1 variant in an age-dependent manner. In addition, they support the view that the PS1 overexpression promotes synaptic dysfunction in an Abeta-independent manner and underline the crucial role of PS1 during both normal and pathological aging.

Immune responses of IL-5 transgenic mice to parasites and aeroallergens

Eosinophils have long been thought to be effectors of immunity to helminths but have also been implicated in the pathogenesis of asthma. Patterns of cytokine production in the host may influence the pathogenesis of these diseases by regulating the activities of eosinophils and other components of the immune response. Mice which constitutively over-express IL-5 have profound and life-long eosinophilia in a restricted number of tissues. Although eosinophils from IL-5 transgenics are functionally competent for a number of parameters considered to be important in inflammation, untreated animals are overtly normal and free of disease. In addition, the responses of these animals when exposed to aeroallergens and helminths present a number of apparent paradoxes. Eosinophil accumulation in tissues adjacent to major airways is rapid and extensive in transgenics exposed to the aeroallergen, but even after treatment with antigen over many months these mice show no evidence of respiratory distress or pathology. Helminth-infected IL-5 transgenics and their non-transgenic littermates develop similar inflammatory responses at mucosal sites and are comparable for a number of T cell and antibody responses, but they differ considerably in their ability to clear some parasite species. The life-cycle of Nippostrongylus brasiliensis is significantly inhibited in IL-5 transgenics, but that of Toxocara canis is not. Our results also suggest that eosinophilia and/or over-expression of IL-5 may actually impair host resistance to Schistosoma mansoni and Trichinella spiralis. The pathogenesis of diseases in which eosinophils are involved may therefore be more complex than previously thought.

Activation of the heat shock response in plants by chlorophenols: transgenic Physcomitrella patens as a sensitive biosensor for organic pollutants.

The ability to detect early molecular responses to various chemicals is central to the understanding of biological impact of pollutants in a context of varying environmental cues. To monitor stress responses in a model plant, we used transgenic moss Physcomitrella patens expressing the beta-glucuronidase reporter (GUS) under the control of the stress-inducible promoter hsp17.3B. Following exposure to pollutants from the dye and paper industry, GUS activity was measured by monitoring a fluorescent product. Chlorophenols, heavy metals and sulphonated anthraquinones were found to specifically activate the hsp17.3B promoter (within hours) in correlation with long-term toxicity effects (within days). At mildly elevated physiological temperatures, the chemical activation of this promoter was strongly amplified, which considerably increased the sensitivity of the bioassay. Together with the activation of hsp17.3B promoter, chlorophenols induced endogenous chaperones that transiently protected a recombinant thermolabile luciferase (LUC) from severe heat denaturation. This sensitive bioassay provides an early warning molecular sensor to industrial pollutants under varying environments, in anticipation to long-term toxic effects in plants. Because of the strong cross-talk between abiotic and chemical stresses that we find, this P. patens line is more likely to serve as a direct toxicity bioassay for pollutants combined with environmental cues, than as an indicator of absolute toxicity thresholds for various pollutants. It is also a powerful tool to study the role of heat shock proteins (HSPs) in plants exposed to combined chemical and environmental stresses.

Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites.

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice.

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.

Selective absence of CD8+ TCRalpha beta+ intestinal epithelial cells in transgenic mice expressing beta2-microglobulin-associated ligands exclusively on thymic cortical epithelium.

Whereas interactions between the TCRalpha beta and self MHC:peptide complexes are clearly required for positive selection of mature CD4(+) and CD8(+) T cells during intrathymic development, the role of self or foreign ligands in maintaining the peripheral T cell repertoire is still controversial. In this report we have utilized keratin 14-beta2-microglobulin (K14-beta2m)-transgenic mice expressing beta2m-associated ligands exclusively on thymic cortical epithelial cells to address the possible influence of TCR:ligand interactions in peripheral CD8(+) T cell homeostasis. Our data indicate that CD8(+) T cells in peripheral lymphoid tissues are present in normal numbers in the absence of self MHC class I:peptide ligands. Surprisingly, however, steady state homeostasis of CD8(+) T cells in the intestinal epithelium is severely affected by the absence of beta2m-associated ligands. Indeed TCRalpha beta(+) IEL subsets expressing CD8alpha beta or CD8alpha alpha are both dramatically reduced in K14-beta2m mice, suggesting that the development, survival or expansion of CD8(+) IEL depends upon interaction of the TCR with MHC class I:peptide or other beta2m-associated ligands elsewhere than on thymic cortical epithelium. Collectively, our data reveal an unexpected difference in the regulation of CD8(+) T cell homeostasis by beta2m-associated ligands in the intestine as compared to peripheral lymphoid organs.

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.

Perspectives in the control of infectious diseases by transgenic mosquitoes in the post-genomic era: a review

Arthropod-borne diseases caused by a variety of microorganisms such as dengue virus and malaria parasites afflict billions of people worldwide imposing major economic and social burdens. Despite many efforts, vaccines against diseases transmitted by mosquitoes, with the exception of yellow fever, are not available. Control of such infectious pathogens is mainly performed by vector management and treatment of affected individuals with drugs. However, the numbers of insecticide-resistant insects and drug-resistant parasites are increasing. Therefore, inspired in recent years by a lot of new data produced by genomics and post-genomics research, several scientific groups have been working on different strategies to control infectious arthropod-borne diseases. This review focuses on recent advances and perspectives towards construction of transgenic mosquitoes refractory to malaria parasites and dengue virus transmission.

Targeted expression of human CD1d in transgenic mice reveals independent roles for thymocytes and thymic APCs in positive and negative selection of Valpha14i NKT cells.

CD1d-dependent invariant Valpha14 (Valpha14i) NKT cells are innate T lymphocytes expressing a conserved semi-invariant TCR, consisting, in mice, of the invariant Valpha14-Jalpha18 TCR alpha-chain paired mostly with Vbeta8.2 and Vbeta7. The cellular requirements for thymic positive and negative selection of Valpha14i NKT cells are only partially understood. Therefore, we generated transgenic mice expressing human CD1d (hCD1d) either on thymocytes, mainly CD4+ CD8+ double positive, or on APCs, the cells implicated in the selection of Valpha14i NKT cells. In the absence of the endogenous mouse CD1d (mCD1d), the expression of hCD1d on thymocytes, but not on APCs, was sufficient to select Valpha14i NKT cells that proved functional when activated ex vivo with the Ag alpha-galactosyl ceramide. Valpha14i NKT cells selected by hCD1d on thymocytes, however, attained lower numbers than in control mice and expressed essentially Vbeta8.2. The low number of Vbeta8.2+ Valpha14i NKT cells selected by hCD1d on thymocytes was not reversed by the concomitant expression of mCD1d, which, instead, restored the development of Vbeta7+ Valpha14i NKT cells. Vbeta8.2+, but not Vbeta7+, NKT cell development was impaired in mice expressing both hCD1d on APCs and mCD1d. Taken together, our data reveal that selective CD1d expression by thymocytes is sufficient for positive selection of functional Valpha14i NKT cells and that both thymocytes and APCs may independently mediate negative selection.

Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up to 4% of their fresh weight.

Transgenic Arabidopsis thaliana (L.) Heynh. plants expressing the three enzymes encoding the biosynthetic route to polyhydroxybutyrate (PHB) are described. These plants accumulated more than 4% of their fresh weight (approximately 40% of their dry weight) in the form of PHB in leaf chloroplasts. These very high producers were obtained and identified following a novel strategy consisting of a rapid GC-MS analysis of a large number of transgenic Arabidopsis plants generated using a triple construct, thus allowing the parallel transfer of all three genes necessary for PHB synthesis in a single transformation event. The level of PHB produced was 4-fold greater than previously published values, thus demonstrating the large potential of plants to produce this renewable resource. However, the high levels of the polymer produced had severe effects on both plant development and metabolism. Stunted growth and a loss of fertility were observed in the high-producing lines. Analysis of the metabolite composition of these lines using a GC-MS method that we have newly developed showed that the accumulation of high levels of PHB was not accompanied by an appreciable change in either the composition or the amount of fatty acids. Substantial changes were, however, observed in the levels of various organic acids, amino acids, sugars and sugar alcohols.

Mice Transgenic For Human Dominant Mutations Of The GUCY2D Gene

Purpose: Animal models are essential to study pathological mechanisms and to test new therapeutic strategies. Many mouse models mimic human rod loss but only a limited number simulate cone dystrophies. The importance of cone function for human vision highlights the need to engineer a model for cone degeneration. An approach of lentiviral-directed transgenesis was tested in mice to express a dominant mutant gene described in a human cone dystrophy.Methods: Lentiviral vectors (LV) encoding either hrGFPII or the human double mutant GUCY2DE837D/R838S cDNA under the control of a region of the pig arrestin-3 promoter (Arr3) were produced and used for lentiviral-derived transgenesis. PCR-genotyping determined the transgenic mouse ratio. The expression of GFP was then analyzed both in vivo and by immunohistochemistry in Arr3-GFPII mice. Functional analysis was performed by ERG at 5, 9, 16 and 24 weeks for Arr3-GUCY2DE837D/R838S mice. Mice were sacrificed at 10 months of age for both histological analysis and RNA extraction.Results: While all the newborns from the transgenesis using the LV-Arr3-GFPII were transgenic, one third of the newborns from the LV-Arr3-GUCY2DE837D/R838S transgenesis were positive. Expression of GFPII was demonstrated by in vivo imaging, while expression of the mutant GUCY2D transcript was detetected using RT-PCR. No severe alteration of the functional response was observed up to 24 weeks of age in the transgenic mice. No obvious modification of the retinal morphology was identified either.Conclusions: Lentiviral-directed transgenesis is a rapid and straightforward method to engineer transgenic mice. Protein expression can be specifically targeted to the retina and thus could help to study the effect of expression of dominant mutant proteins. In our case, Arr3-GUCY2DE837D/R838S mice have a less severe phenotype than that described for human patients. Further analyses are required to understand this difference but several modifications of the expression cassette might also help to increase the expression of the mutant protein and reinforce the phenotype. Interestingly, the same construct is less effective in mouse versus pig retina (see Arsenijevic et al. ARVO 2011 abstract).

Genotypic features of lentivirus transgenic mice.

Lentivector-mediated transgenesis is increasingly used, whether for basic studies as an alternative to pronuclear injection of naked DNA or to test candidate gene therapy vectors. In an effort to characterize the genetic features of this approach, we first measured the frequency of germ line transmission of individual proviruses established by infection of fertilized mouse oocytes. Seventy integrants from 11 founder (G0) mice were passed to 111 first generation (G1) pups, for a total of 255 events corresponding to an average rate of transmission of 44%. This implies that integration had most often occurred at the one- or two-cell stage and that the degree of genotypic mosaicism in G0 mice obtained through this approach is generally minimal. Transmission analysis of eight individual proviruses in 13 G2 mice obtained by a G0-G1 cross revealed only 8% of proviral homozygosity, significantly below the 25% expected from purely Mendelian transmission, suggesting counter-selection due to interference with the functions of targeted loci. Mapping of 239 proviral integration sites in 49 founder animals revealed that about 60% resided within annotated genes, with a marked tendency for clustering in the middle of the transcribed region, and that integration was not influenced by the transcriptional orientation. Transcript levels of a set of arbitrarily chosen target genes were significantly higher in two-cell embryos than in embryonic stem cells or adult somatic cells, suggesting that, as previously noted in other settings, lentiviral vectors integrate preferentially into regions of the genome that are transcriptionally active or poised for activation.

New insights into dendritic cell biology and histiocytosis through a transgenic tumor model

SUMMARY The effective development of an immune response depends on the careful interplay and the regulation between innate and adaptive immunity. As the dendritic cells (DCs) are equipped with many receptors, such as Toll-like receptors, which can detect the presence of infection by recognizing different component of bacteria, fungi and even viruses, they are the among the first cells to respond to the infection. Upon pathogen challenge, the DCs interpret the innate system activation as a maturation signal, resulting in the migration of the DCS to a draining lymph node site. There, activated DCs present efficiently antigens to naïve T cells, which are in turn activated and initiate adaptive immunity. Therefore, DCs are the main connectors between innate and adaptive immune systems. In addition to be the most efficient antigen- presenting cells, DCs play a central role in the regulation of immune responses and immune tolerance. Despite extensive research, many aspects related to DC biology are still unsolved and/or controversial. The low frequency of DCs in vivo often hamper study of DC biology and in vitro-derived DCs are not suited to address certain questions, such as the development of DC. We sought of transforming in vivo the DCs through the specific expression of an oncogene, in order to obtain unlimited numbers of these cells. To achieve this goal, transgenic mouse lines expressing the SV40 Large T oncogene under the control of the CD1 1 c promoter were generated. These transgenic mice are healthy until the age of three to four months without alterations in the DC biology. Thereafter transgenic mice develop a fatal disease that shows features of a human pathology, named histiocytosis, involving DCs. We demonstrate that the disease development in the transgenic mice correlates with a massive accumulation of transformed DCs in the affected organs. Importantly, transformed DCs are immature and fully conserve their capacity to mature in antigen presenting cells. We observe hyperproliferation of transformed DCs only in the sick transgenic mice. Surprisingly, transformed DCs do not proliferate in vitro, but transfer of the transformed DCs into immunodeficient or tolerant host leads to tumor formation. Altoghether, the transgenic mouse lines we have generated represent a valuable tumor model for human histiocytosis, and provide excellent tools to study DC biology. RESUME Le développement d'une réponse immunitaire efficace dépend d'une minutieuse interaction et régulation entre l'immunité innée et adaptative. Comme les cellules dendritiques (DCs) sont équipées de nombreux récepteurs, tels que les récepteurs Toll-like, qui peuvent détecter la présence d'une infection en reconnaissant différents composants bactériens, issus de champignons ou même viraux, elles sont parmi les premières cellules à répondre à l'infection. Suite à la stimulation induite par le pathogène, les DCs interprètent l'activation du système immunitaire inné comme un signal de maturation, résultant dans la migration des DCs vers le ganglion drainant le site d'infection. Là, les DCs actives présentent efficacement des antigènes aux cellules T, qui sont à leur tour activées et initient les systèmes d'immunité adaptative. Ainsi, les DCs forment le lien principal entre les réponses immunitaires innées et adaptatives. En plus d'être les cellules présentatrices d'antigènes les plus efficaces, les DCs jouent un rôle central dans la régulation du système immunitaire et dans le phénomène de tolérance. Malgré des recherches intensives, de nombreux aspects liés à la biologie des DCs sont encore irrésolus et/ou controversés. La faible fréquence des DCs in vivo gêne souvent l'étude de la biologie de ces cellules et les DCs dérivées in vitro ne sont pas adéquates pour adresser certaines questions, telles que le développement des DCs. Afin d'obtenir des quantités illimitées de DCs, nous avons songé à transformer in vivo les DC grâce à l'expression spécifique d'un oncogène. Afin d'atteindre ce but, nous avons généré des lignées de souris transgéniques qui expriment l'oncogène SV40 Large T sous le contrôle du promoter CD1 le. Ces souris transgéniques sont saines jusqu'à l'âge de trois à quatre mois et ne présentent pas d'altération dans la biologie des DCs. Ensuite, les souris transgéniques développent une maladie présentant les traits caractéristiques d'une pathologie humaine nommée histiocytose, qui implique les DCs. Nous montrons que le développement de cette maladie corrèle avec une accumulation massive des DCs transformées dans les organes touchés. De plus, les DCs transformées sont immatures et conservent leur capacité à différencier en cellules présentatrices d'antigène. Nous observons une hyper-prolifération des DCs transformées seulement dans les souris transgéniques malades. Etonnament, les DC transformées ne prolifèrent pas in vitro, par contre, le transfert des DCs transformées dans des hôtes immuno-déficients ou tolérant conduit à la formation de tumeurs. Globalement, les lignées de souris transgéniques que nous avons générées représentent un modèle valide pour l'histiocytose humaine, et de plus, offrent d'excellents outils pour étudier la biologie des DCs.

High-frequency linkage of co-expressing T-DNA in transgenic Arabidopsis thaliana transformed by vacuum-infiltration of Agrobacterium tumefaciens

The efficiency of co-expression and linkage of distinct T-DNAs present in separate Agrobacterium tumefaciens was analysed in Arabidopsis thaliana transformed by the vacuum infiltration method. Co-expression was monitored by the synthesis of three bacterial proteins involved in the production of polyhydroxybutyrate (PHB) in the plastids. Out of 80 kanamycin-resistant transgenic plants analysed, 13 plants were co-transformed with the two distinct T-DNAs and produced PHB. Of those, 7 lines had a kanamycin-resistance segregation ratio consistent with the presence of a single functional insert. Genetic linkage between the distinct T-DNAs was demonstrated for all 13 PHB-producing lines, while physical linkage between the distinct T-DNAs was shown for 12 out of 13 lines. T-DNAs were frequently linked in an inverted orientation about the left borders. Transformation of A. thaliana by the co-infiltration of two A. tumefaciens containing distinct T-DNAs is, thus, an efficient approach for the integration and expression of several transgenes at a single locus. This approach will facilitate the creation and study of novel metabolic pathways requiring the expression of numerous transgenes.