CamKII inhibitors reduce mitotic instability, connexon anomalies and progression of the in vivo behavioral phenotype in transgenic animals expressing a mutated Gjb1 gene

AbstractAmyloid plaques are a hallmark of Alzheimer’s disease (AD) that develop in its earliest stages. Thus, non-invasive detection of these plaques would be invaluable for diagnosis and the development and monitoring of treatments, but this remains a challenge due to their small size. Here, we investigated the utility of manganese-enhanced MRI (MEMRI) for visualizing plaques in transgenic rodent models of AD across two species: 5xFAD mice and TgF344-AD rats. Animals were given subcutaneous injections of MnCl2 and imaged in vivo using a 9.4 T Bruker scanner. MnCl2 improved signal-to-noise ratio but was not necessary to detect plaques in high-resolution images. Plaques were visible in all transgenic animals and no wild-types, and quantitative susceptibility mapping showed that they were more paramagnetic than the surrounding tissue. This, combined with beta-amyloid and iron staining, indicate that plaque MR visibility in both animal models was driven by plaque size and iron load. Longitudinal relaxation rate mapping revealed increased manganese uptake in brain regions of high plaque burden in transgenic animals compared to their wild-type littermates. This was limited to the rhinencephalon in the TgF344-AD rats, while it was most significantly increased in the cortex of the 5xFAD mice. Alizarin Red staining suggests that manganese bound to plaques in 5xFAD mice but not in TgF344-AD rats. Multi-parametric MEMRI is a simple, viable method for detecting amyloid plaques in rodent models of AD. Manganese-induced signal enhancement can enable higher-resolution imaging, which is key to visualizing these small amyloid deposits. We also present the first in vivo evidence of manganese as a potential targeted contrast agent for imaging plaques in the 5xFAD model of AD.

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Serial analysis of gene expression (SAGE) during porcine embryo development

Reproduction Fertility and Development ◽

10.1071/rd03081 ◽

2004 ◽

Vol 16 (2) ◽

pp. 87 ◽

Cited By ~ 12

Author(s):

Le Ann Blomberg ◽

Kurt A. Zuelke

Keyword(s):

Gene Expression ◽

Functional Genomics ◽

Embryo Development ◽

Molecular Mechanisms ◽

Physiological Role ◽

Transgenic Animals ◽

Specific Gene ◽

Research Strategies

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

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Direct repeats bind the EcR/USP receptor and mediate ecdysteroid responses in Drosophila melanogaster.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.2977 ◽

1996 ◽

Vol 16 (6) ◽

pp. 2977-2986 ◽

Cited By ~ 70

Author(s):

C Antoniewski ◽

B Mugat ◽

F Delbac ◽

J A Lepesant

Keyword(s):

Fat Body ◽

Cell Transformation ◽

Direct Repeat ◽

Transgenic Animals ◽

Transcriptional Level ◽

Regulatory Sequences ◽

Direct Repeats ◽

Fbp1 Gene

The steroid hormone 20-hydroxyecdysone plays a key role in the induction and modulation of morphogenetic events throughout Drosophila development. Previous studies have shown that a heterodimeric nuclear receptor composed of the EcR and USP proteins mediates the action of the hormone at the transcriptional through binding to palindromic ecdysteroid mediates the action of the hormone at the transcriptional level through binding to palindromic ecdysteroid response elements (EcREs) such as those present in the promoter of the hsp27 gene or the fat body-specific enhancer of the Fbp1 gene. We show that in addition to palindromic EcREs, the EcR/USP heterodimer can bind in vitro with various affinities to direct repetitions of the motif AGGTCA separated by 1 to 5 nucleotides (DR1 to DR5), which are known to be target sites for vertebrate nuclear receptors. At variance with the receptors, EcR/USP was also found to bind to a DR0 direct repeat with no intervening nucleotide. In cell transformation assays, direct repeats DR0 to DR5 alone can render the minimum viral tk or Drosophila Fbp1 promoter responsive to 20-hydroxyecdysone, as does the palindromic hsp27 EcRE. In a transgenic assay, however, neither the palindromic hsp27 element nor direct repeat DR3 alone can make the Fbp1 minimal promoter responsive to premetamorphic ecdysteroid peaks. In contrast, DR0 and DR3 elements, when substituted for the natural palindromic EcRE in the context of the Fbp1 enhancer, can drive a strong fat body-specific ecdysteroid response in transgenic animals. These results demonstrate that directly repeated EcR/USP binding sites are as effective as palindromic EcREs in vivo. They also provide evidence that additional flanking regulatory sequences are crucially required to potentiate the hormonal response mediated by both types of elements and specify its spatial and temporal pattern.

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The c-myc proto-oncogene regulates cardiac development in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.10.7.3709 ◽

1990 ◽

Vol 10 (7) ◽

pp. 3709-3716 ◽

Cited By ~ 128

Author(s):

T Jackson ◽

M F Allard ◽

C M Sreenan ◽

L K Doss ◽

S P Bishop ◽

...

Keyword(s):

Transgenic Mice ◽

Mrna Expression ◽

Cardiac Myocyte ◽

Cellular Proliferation ◽

Cardiac Development ◽

Constitutive Expression ◽

Transgenic Animals ◽

Hypertrophic Growth ◽

Myocyte Proliferation

During the maturation of the cardiac myocyte, a transition occurs from hyperplastic to hypertrophic growth. The factors that control this transition in the developing heart are unknown. Proto-oncogenes such as c-myc have been implicated in the regulation of cellular proliferation and differentiation, and in the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc can influence myocyte proliferation or differentiation, we examined the in vivo effect of increasing c-myc expression during embryogenesis and of preventing the decrease in c-myc mRNA expression that normally occurs during cardiac development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. In these transgenic mice, increased c-myc mRNA expression was found to be associated with both atrial and ventricular enlargement. This increase in cardiac mass was secondary to myocyte hyperplasia, with the transgenic hearts containing more than twice as many myocytes as did nontransgenic hearts. The results suggest that in the transgenic animals there is additional hyperplastic growth during fetal development. However, this additional proliferative growth is not reflected in abnormal myocyte maturation, as assessed by the expression of the cardiac and skeletal isoforms of alpha-actin. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth and suggest a regulatory role for this proto-oncogene in cardiac myogenesis.

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Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽

10.1534/genetics.109.106567 ◽

2009 ◽

Vol 183 (3) ◽

pp. 1165-1173 ◽

Cited By ~ 34

Author(s):

Shu Kondo ◽

Matthew Booker ◽

Norbert Perrimon

Keyword(s):

Cell Culture ◽

Drosophila Melanogaster ◽

Large Scale ◽

Transgenic Animals ◽

Selection Marker ◽

Bacterial Artificial Chromosomes ◽

Loss Of Function ◽

Artificial Chromosomes ◽

Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

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MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816656115 ◽

2018 ◽

Vol 116 (1) ◽

pp. 303-312 ◽

Cited By ~ 24

Author(s):

Erol C. Bayraktar ◽

Lou Baudrier ◽

Ceren Özerdem ◽

Caroline A. Lewis ◽

Sze Ham Chan ◽

...

Keyword(s):

Metabolite Profiling ◽

Cultured Cells ◽

Transgenic Animals ◽

Cell Types ◽

Tissue Level ◽

Specific Cell ◽

Mammalian Tissues ◽

Cell Type Specific ◽

Untargeted Metabolite Profiling

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.

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Overexpression of the Matrix Metalloproteinase Matrilysin Results in Premature Mammary Gland Differentiation and Male Infertility

Molecular Biology of the Cell ◽

10.1091/mbc.9.2.421 ◽

1998 ◽

Vol 9 (2) ◽

pp. 421-435 ◽

Cited By ~ 36

Author(s):

Laura A. Rudolph-Owen ◽

Paul Cannon ◽

Lynn M. Matrisian

Keyword(s):

Matrix Metalloproteinase ◽

Transgenic Animals ◽

Reproductive Organs ◽

Mammary Development ◽

Seminiferous Tubules ◽

Interstitial Tissue ◽

Wild Type ◽

The Matrix

To examine the role of matrilysin (MAT), an epithelial cell-specific matrix metalloproteinase, in the normal development and function of reproductive tissues, we generated transgenic animals that overexpress MAT in several reproductive organs. Three distinct forms of human MAT (wild-type, active, and inactive) were placed under the control of the murine mammary tumor virus promoter/enhancer. Although wild-type, active, and inactive forms of the human MAT protein could be produced in an in vitro culture system, mutations of the MAT cDNA significantly decreased the efficiency with which the MAT protein was produced in vivo. Therefore, animals carrying the wild-type MAT transgene that expressed high levels of human MAT in vivo were further examined. Mammary glands from female transgenic animals were morphologically normal throughout mammary development, but displayed an increased ability to produce β-casein protein in virgin animals. In addition, beginning at approximately 8 mo of age, the testes of male transgenic animals became disorganized with apparent disintegration of interstitial tissue that normally surrounds the seminiferous tubules. The disruption of testis morphology was concurrent with the onset of infertility. These results suggest that overexpression of the matrix-degrading enzyme MAT alters the integrity of the extracellular matrix and thereby induces cellular differentiation and cellular destruction in a tissue-specific manner.

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Highly efficient lentiviral-mediated human cytokine transgenesis on the NOD/scid background

Blood ◽

10.1182/blood-2003-07-2298 ◽

2004 ◽

Vol 103 (2) ◽

pp. 580-582 ◽

Cited By ~ 13

Author(s):

Isabel Punzon ◽

Luis M. Criado ◽

Alfredo Serrano ◽

Fernando Serrano ◽

Antonio Bernad

Keyword(s):

Transgenic Mice ◽

Lentiviral Vector ◽

Cell Lineage ◽

Transgenic Animals ◽

Hematopoietic Tissue ◽

Nonobese Diabetic ◽

Human Blood Cell ◽

Macrophage Colony ◽

Human Cytokine

Abstract Human neo-organ formation from stem cells can only be assayed by in vivo xenotransplantation. The human nonobese diabetic–severe combined immunodeficient (HuNOD/scid) CD34+ cell transplantation is a model that allows examination of hematopoietic tissue formation, although human hematopoietic cell maturation is abortive. Conventional humanization of the cytokine microenvironment has depended on generation of human cytokine-transgenic mice in strains appropriate for conventional plasmid microinjection, followed by backcrossing, a costly and time-consuming approach. Lentiviral vector infection of single-cell embryos was recently reported to produce transgenic animals. Using this approach, we have generated direct human granulocyte-macrophage colony-stimulating factor (hGM-CSF) transgenic mice from lentivirus-microinjected NOD/scid embryos, with 68% efficiency and 100% penetrance; this allowed us to obtain NOD/scid transgenic mice with considerable savings of resources. This powerful technique should assist in producing novel mouse models for the study of human blood cell lineage development and other human neo-organs from stem cell xenotransplantation for which a similar “humanization” rationale may be required.

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MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

10.1101/425454 ◽

2018 ◽

Author(s):

Erol Can Bayraktar ◽

Lou Baudrier ◽

Ceren Özerdem ◽

Caroline A. Lewis ◽

Sze Ham Chan ◽

...

Keyword(s):

Metabolite Profiling ◽

Cultured Cells ◽

Transgenic Animals ◽

Cell Types ◽

Tissue Level ◽

Specific Cell ◽

Mammalian Tissues ◽

Cell Type Specific ◽

Untargeted Metabolite Profiling

ABSTRACTMitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell-types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially-localized 3XHA epitope-tag (“MITO-Tag”) for the fast isolation of mitochondria from cultured cells to now generate “MITO-Tag Mice.” Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology and our strategy should be generally applicable for studying other mammalian organelles in specific cell-types in vivo.

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Dexibuprofen ameliorates peripheral and central risk factors associated with Alzheimer’s disease in metabolically stressed APPswe/PS1dE9 mice

10.21203/rs.3.rs-415246/v1 ◽

2021 ◽

Author(s):

Miren Ettcheto ◽

Elena Sánchez-Lopez ◽

Amanda Cano ◽

Marina Carrasco ◽

Katherine Herrera ◽

...

Keyword(s):

Cognitive Decline ◽

Combined Therapy ◽

Chronic Administration ◽

Transgenic Animals ◽

Amyloid Plaque ◽

Tolerance Test ◽

Metabolic Effects ◽

Metabolic Disturbances ◽

Insulin Tolerance

Abstract Background: Several studies stablished a relationship between metabolic disturbances and Alzheimer´s disease (AD) where inflammation plays a pivotal role. However, mechanisms involved still remain unclear. In the present study, we aimed to evaluate central and peripheral effects of dexibuprofen (DXI) in the progression of AD in APPswe/PS1dE9 (APP/PS1) female mice, a familial AD model, fed with high fat diet (HFD). Animals were fed either with conventional chow or with HFD, from their weaning until their sacrifice, at 6 months. Moreover, mice were divided into subgroups to which were administered drinking water or water supplemented with DXI (20mg·kg-1·d-1) for 3 months. Before sacrifice, body weight, intraperitoneal glucose and insulin tolerance test (IP-ITT) were performed to evaluate peripheral parameters and also behavioral tests to determine cognitive decline. Moreover, molecular studies such as Western blot and RT-PCR were carried out in liver to confirm metabolic effects and in hippocampus to analyze several pathways considered hallmarks in AD. Results: Our studies demonstrate that DXI improved metabolic alterations observed in transgenic animals fed with HFD in vivo, data in accordance with those obtained at molecular level. Moreover, an improvement of cognitive decline and neuroinflammation among other pathways associated with AD were observed such as beta-amyloid plaque accumulation and unfolded protein response.Conclusions: Collectively, evidence suggest that chronic administration of DXI prevents the progression of AD through the regulation of inflammation which contribute to improve hallmarks of this pathology. Thus, this compound could constitute a novel therapeutic approach in the treatment of AD in a combined therapy.

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