scholarly journals Redirection of tumor metastasis by expression of E-selectin in vivo.

1996 ◽  
Vol 183 (2) ◽  
pp. 581-587 ◽  
Author(s):  
L Biancone ◽  
M Araki ◽  
K Araki ◽  
P Vassalli ◽  
I Stamenkovic
Keyword(s):  
Transgenic Mice ◽  
Direct Evidence ◽  
Cell Attachment ◽  
Liver Tumors ◽  
Leukocyte Adhesion ◽  
Critical Role ◽  
Promote Tumor Cell ◽  
Histologic Pattern ◽  
Alpha 1 Antitrypsin

The selectin class of adhesion molecules plays a critical role in facilitating leukocyte adhesion to and subsequent transmigration of endothelium. On this basis, selectins have been suggested to promote tumor cell attachment to endothelium, thereby facilitating metastasis of certain types of tumors, although direct evidence for such a role is lacking. To explore this hypothesis, two sets of transgenic mice were developed: TgnES, which constitutively expresses cell surface E-selectin in all tissues, under the control of the beta-actin promoter; and TgnEsol, which expresses truncated, soluble E-selectin in the liver, under the control of the alpha 1 antitrypsin promoter. B16F10 melanoma cells were stably transfected with alpha(1,3/1,4) fucosyltransferase-specific cDNA (B16F10ft), allowing them to express E-selectin ligands or with hygromycin resistance selection vector only B16F10hygro). Normal mice injected with B16F10ft and B16F10hygro and transgenic mice injected with B16F10hygro developed lung tumors exclusively. In contrast, TgnES mice injected with B16F10ft cells developed massive infiltrating liver tumors. B16F10ft cells injected into TgnEsol mice also formed liver tumors, but these grew more slowly, with a well-delineated, noninfiltrating distinct histologic pattern. These observations provide direct evidence that expression of E-selectin can redirect metastasis of tumor cells expressing appropriate ligands in vivo.

Dysregulation of MMSET Is Tumorigenic In-Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 74-74 ◽  
Author(s):  
Marta Chesi ◽  
Kruti Naik ◽  
Davide F. Robbiani ◽  
Maurizio Affer ◽  
Helen D. Nickerson ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Transgenic Mice ◽  
Cell Lines ◽  
Germinal Center ◽  
Somatic Hypermutation ◽  
Critical Role ◽  
Lymphoid Cells ◽  
Proximal Promoter

Abstract Approximately 15% of multiple myeloma (MM) is characterized by a t(4;14) translocation that causes the simultaneous dysregulation of MMSET on der(4) and fibroblast growth factor receptor 3 gene (FGFR3) on der(14). We reported several lines of evidence indicating a role for FGFR3 in myeloma tumorigenesis. First, activated FGFR3 is an oncogene capable of transforming fibroblasts. Second, FGFR3 activating mutations are acquired by MM cells during tumor progression. Third, targeted inhibition of FGFR3 leads to terminal differentiation and apoptosis in two t(4;14) MM cell lines. However, expression of FGFR3, but never of MMSET, is lost in about 25% of t(4;14) MM. Therefore, the overexpression of MMSET in all MM tumors with a t(4;14) translocation, and its homology to MLL, the oncogene on 11q23 translocated in acute leukemia suggest a critical role for MMSET in MM. To determine whether MMSET is an oncogene in vivo, we have generated transgenic mice in which MMSET expression in driven in lymphocytes by the lck proximal promoter juxtaposed to the Emu enhancer. Using the same expression vector we and others have obtained specific, high levels of transgene expression in B and T cells from spleen, bone marrow and thymus. Four transgenic lines were generated and although we detected MMSET expression in T cells in each of them, unexpectedly no expression in B cells was seen. This is consistent with our inability to ectopically express MMSET in B cell lines. Nevertheless B lymphoid tumors expressing MMSET developed at 23 month of age in each line (18/51 mice). Only 1/19 wild type matching control mice developed a splenomegaly. By Southern blot, monoclonal rearrangements of IgH, IgL and TCR β were detected within the same tumor population. In conclusion, this is the first report that MMSET is an oncogene capable of transforming lymphoid cells in an animal model. We are currently crossing these mice with FGFR3 transgenic mice to assess cooperation between these two oncogenes in tumorigenesis. Obviously a more restricted expression of MMSET in germinal center cells is required to investigate the role of MMSET in MM. Therefore, as we have done for c-myc, we are generating new transgenic mice in which MMSET expression will be activated sporadically in germinal center B cells by somatic hypermutation.

scholarly journals Generation of Fad2 and Fad3 transgenic mice that produce n-6 and n-3 polyunsaturated fatty acids

Open Biology ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 190140 ◽  
Author(s):  
Lishuang Song ◽  
Lei Yang ◽  
Jiapeng Wang ◽  
Xuefei Liu ◽  
Lige Bai ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Transgenic Mice ◽  
Polyunsaturated Fatty Acids ◽  
Critical Role ◽  
Fatty Acid Desaturase ◽  
Biosynthetic Pathways ◽  
Wild Type ◽  
Pufa Biosynthesis ◽  
Double Transgenic Mice

Linoleic acid (18 : 2, n-6) and α-linolenic acid (18 : 3, n-3) are polyunsaturated fatty acids (PUFAs), which are essential for mammalian health, development and growth. However, the majority of mammals, including humans, are incapable of synthesizing n-6 and n-3 PUFAs. Mammals must obtain n-6 and n-3 PUFAs from their diet. Fatty acid desaturase (Fad) plays a critical role in plant PUFA biosynthesis. Therefore, we generated plant-derived Fad3 single and Fad2–Fad3 double transgenic mice. Compared with wild-type mice, we found that PUFA levels were greatly increased in the single and double transgenic mice by measuring PUFA levels. Moreover, the concentration of n-6 and n-3 PUFAs in the Fad2–Fad3 double transgenic mice were greater than in the Fad3 single transgenic mice. These results demonstrate that the plant-derived Fad2 and Fad3 genes can be expressed in mammals. To clarify the mechanism for Fad2 and Fad3 genes in transgenic mice, we measured the PUFAs synthesis-related genes. Compared with wild-type mice, these Fad transgenic mice have their own n-3 and n-6 PUFAs biosynthetic pathways. Thus, we have established a simple and efficient method for in vivo synthesis of PUFAs.

scholarly journals Inhibition of autophagy suppresses SARS-CoV-2 replication and ameliorates pneumonia in hACE2 transgenic mice and xenografted human lung tissues

2021 ◽  
Author(s):  
Chao Shang ◽  
Xinyu Zhuang ◽  
He Zhang ◽  
Yiquan Li ◽  
Yilong Zhu ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Transgenic Mice ◽  
Human Lung ◽  
Critical Role ◽  
Suitable Model ◽  
Autophagosome Formation ◽  
Human Lung Tissue ◽  
Global Pandemic

Autophagy is thought to be involved in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. However, how SARS-CoV-2 interferes with the autophagic pathway and whether autophagy contributes to virus infection in vivo is unclear. Here, we identified SARS-CoV-2-triggered autophagy in animal models including the long tailed or crab eating macaque ( Macaca fascicularis ), hACE2 transgenic mice and xenografted human lung tissues. In Vero E6 and Huh-7 cells, SARS-CoV-2 induces autophagosome formation, accompanied by consistent autophagic events, including inhibition of the Akt-mTOR pathway, and activation of the ULK-1-Atg13 and VPS34-VPS15-Beclin1 complexes, but blocks autophagosome-lysosome fusion. Modulation of autophagic elements, including the VPS34 complex and Atg14, but not Atg5, inhibits SARS-CoV-2 replication. Moreover, this study represents the first to demonstrate that the mouse bearing xenografted human lung tissue is a suitable model for SARS-CoV-2 infection and that autophagy inhibition suppresses SARS-CoV-2 replication and ameliorates virus-associated pneumonia in human lung tissues. We also observed a critical role of autophagy in SARS-CoV-2 infection in an hACE2 transgenic mouse model. This study, therefore, gives insights into the mechanisms by which SARS-CoV-2 manipulates autophagosome formation and we suggest that autophagy-inhibiting agents might be useful as therapeutic agents against SARS-CoV-2 infection. IMPORTANCE: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) caused a global pandemic with limited therapeutics. Insights into the virus-host interactions contributes substantially. The novelty of this report is the use of a new animal model: mice xenografted with human lung tissues. Using a combination of the in vitro and in vivo studies, we have provided experimental evidence that induction of autophagy contributes to SARS-CoV-2 infection and improves our understanding of potential therapeutic targets for SARS-CoV-2.

scholarly journals Endothelial TFEB (Transcription Factor EB) Improves Glucose Tolerance via Upregulation of IRS (Insulin Receptor Substrate) 1 and IRS2

2020 ◽  
Author(s):  
Jinjian Sun ◽  
Haocheng Lu ◽  
Wenying Liang ◽  
Guizhen Zhao ◽  
Lu Ren ◽  
...  
Keyword(s):  
Glucose Tolerance ◽  
Transgenic Mice ◽  
High Fat Diet ◽  
Metabolic Diseases ◽  
Critical Role ◽  
Insulin Receptor Substrate ◽  
High Fat ◽  
Transcription Factor Eb

Objective: Vascular endothelial cells (ECs) play a critical role in maintaining vascular homeostasis. Aberrant EC metabolism leads to vascular dysfunction and metabolic diseases. TFEB (transcription factor EB), a master regulator of lysosome biogenesis and autophagy, has protective effects on vascular inflammation and atherosclerosis. However, the role of endothelial TFEB in metabolism remains to be explored. In this study, we sought to investigate the role of endothelial TFEB in glucose metabolism and underlying molecular mechanisms. Approach and Results: To determine whether endothelial TFEB is critical for glucose metabolism in vivo, we utilized EC-selective TFEB knockout and EC-selective TFEB transgenic mice fed a high-fat diet. EC-selective TFEB knockout mice exhibited significantly impaired glucose tolerance compared with control mice. Consistently, EC-selective TFEB transgenic mice showed improved glucose tolerance. In primary human ECs, small interfering RNA-mediated TFEB knockdown blunts Akt (AKT serine/threonine kinase) signaling. Adenovirus-mediated overexpression of TFEB consistently activates Akt and significantly increases glucose uptake in ECs. Mechanistically, TFEB upregulates IRS1 and IRS2 (insulin receptor substrate 1 and 2). TFEB increases IRS2 transcription measured by reporter gene and chromatin immunoprecipitation assays. Furthermore, we found that TFEB increases IRS1 protein via downregulation of microRNAs (miR-335, miR-495, and miR-548o). In vivo, Akt signaling in the skeletal muscle and adipose tissue was significantly impaired in EC-selective TFEB knockout mice and consistently improved in EC-selective TFEB transgenic mice on high-fat diet. Conclusions: Our data revealed a critical role of TFEB in endothelial metabolism and suggest that TFEB constitutes a potential molecular target for the treatment of vascular and metabolic diseases.

Fibroblast Growth Factor 2 Induces Increased Calvarial Osteoblast Proliferation and Cranial Suture Fusion

2002 ◽  
Vol 39 (5) ◽  
pp. 487-496 ◽  
Author(s):  
Amr M. Moursi ◽  
Phillip L. Winnard ◽  
Alissa V. Winnard ◽  
John M. Rubenstrunk ◽  
Mark P. Mooney
Keyword(s):  
Growth Factors ◽  
Organ Culture ◽  
Cell Morphology ◽  
Cell Attachment ◽  
Critical Role ◽  
Blue Staining ◽  
Fibroblast Growth ◽  
Cranial Suture ◽  
Suture Fusion

Objective: Craniosynostosis has been associated with fibroblast growth factors (FGFs) and their receptors. The purpose of this study was to quantitatively determine the effect of FGF2 on rat calvarial osteoblasts and a rat cranial suture formation model. Design: Fetal rat calvarial osteoblasts were cultured with and without FGF2. Cell attachment and proliferation was determined by alamarBlue dye assay and cell morphology by toluidine-blue staining. In rat calvarial organ culture, postnatal day 15 rat calvariae with dura mater were placed in serum-free media with and without FGF2. A unique quantitative analysis of suture fusion was developed by obtaining measurements of suture bridging in histological serial sections at progressive stages of fusion. Results: Attachment for cells treated with FGF2 was similar to control. In contrast, proliferation was higher for cells treated with FGF2 while maintaining an osteoblastic morphology. After 5 days in organ culture, FGF2-treated posterior frontal sutures showed a dramatic increase in fusion, compared with untreated controls. This increased fusion was maintained throughout days 7 and 10 in culture. Also, fusion was enhanced on the dural side of the suture, as is normally observed in vivo, and the normal tissue architecture was maintained. Conclusions: These results indicate that FGF2 can promote rat osteoblast attachment and normal cell morphology as well as induce cell proliferation. In calvarial organ culture, FGF2 treatment produced an enhanced suture fusion. These results provide further support for a critical role for FGF2 in cranial suture development. These studies also present a new quantitative approach to evaluating the effect of suture-perturbing growth factors on cranial suture fusion.

scholarly journals Constitutively active ezrin increases membrane tension, slows migration, and impedes endothelial transmigration of lymphocytes in vivo in mice

Blood ◽  
2012 ◽  
Vol 119 (2) ◽  
pp. 445-453 ◽  
Author(s):  
Yin Liu ◽  
Natalya V. Belkina ◽  
Chung Park ◽  
Raj Nambiar ◽  
Scott M. Loughhead ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Lymph Nodes ◽  
Shape Change ◽  
Critical Role ◽  
Membrane Tension ◽  
Cortical Actin ◽  
Lymphocyte Migration ◽  
Lymphocyte Membrane

ERM (ezrin, radixin moesin) proteins in lymphocytes link cortical actin to plasma membrane, which is regulated in part by ERM protein phosphorylation. To assess whether phosphorylation of ERM proteins regulates lymphocyte migration and membrane tension, we generated transgenic mice whose T-lymphocytes express low levels of ezrin phosphomimetic protein (T567E). In these mice, T-cell number in lymph nodes was reduced by 27%. Lymphocyte migration rate in vitro and in vivo in lymph nodes decreased by 18% to 47%. Lymphocyte membrane tension increased by 71%. Investigations of other possible underlying mechanisms revealed impaired chemokine-induced shape change/lamellipod extension and increased integrin-mediated adhesion. Notably, lymphocyte homing to lymph nodes was decreased by 30%. Unlike most described homing defects, there was not impaired rolling or sticking to lymph node vascular endothelium but rather decreased migration across that endothelium. Moreover, decreased numbers of transgenic T cells in efferent lymph suggested defective egress. These studies confirm the critical role of ERM dephosphorylation in regulating lymphocyte migration and transmigration. Of particular note, they identify phospho-ERM as the first described regulator of lymphocyte membrane tension, whose increase probably contributes to the multiple defects observed in the ezrin T567E transgenic mice.

REGULATION OF HUNTINGTIN PALITOYLATION AND ITS ROLE IN HUNTINGTON DISEASE

2008 ◽  
Vol 31 (4) ◽  
pp. 26
Author(s):  
Fiona BJ Young ◽  
Deborah Yu Deng ◽  
Roshni R Singaraja ◽  
Michael R Hayden
Keyword(s):  
Transgenic Mice ◽  
Huntington Disease ◽  
Health Research ◽  
Psychiatric Symptoms ◽  
Ex Vivo ◽  
Critical Role ◽  
Junior Trainee ◽  
Bac Transgenic Mice

Huntington Disease(HD) is an inherited and ultimately fatal neurodegenerative disease demonstrating both neurological and psychiatric symptoms. The protein huntingtin (htt) undergoes many post-translational modifications, such as phosphorylation, palmitoylation, and proteolysis. Palmitoylation, the process by which a 16-carbon fatty acid forms a thioester bond with cysteine residues, is a reversible modification known to influence protein trafficking and function. Huntingtin Interacting Protein 14 (HIP14) was identified as a major palmitoyl acyl transferase (PAT) that interacts robustly with wild-type htt, but has significantly reduced interaction with mutant polyglutamine expanded htt. HIP14is a major PAT for htt and palmitoylation of mutant htt by HIP14 is significantly reduced. Down regulation of HIP14 by siRNA in vitro results in increased cell death in neurons, whereas co-transfection with htt and HIP14 results in enhanced palmitoylation and reduction in number of inclusions. Our laboratory has developed a HIP14 knockout mouse (HIP14-/-), which demonstrates many HD-like features similar to those seen in the YAC128 transgenic mouse model of HD. Notably, these mice demonstrate a much earlier and more severe phenotype as compared to the YAC128 mice, suggesting a critical role for HIP14 in HD pathogenesis. The overarching goal of these studies is to explore the role of HIP14 and palmitoylation in the pathogenesis of HD using in vitro, in vivo, and ex vivo methods. A human Bacterial Artificial Chromosome (BAC) containing HIP14 was identified, prepared, and submitted for microinjection, and we have now generated HIP14-overexpressing transgenic mice. These mice are currently undergoing preliminary analyses, and a subset of founders will be selected to undergo further characterization. These human HIP14 BAC transgenic mice will represent the first mammalian model of PAT overexpression, and will significantly further our understanding of PAT activity in vivo. The YAC transgenic model for HD, developed previously in our laboratory, recapitulates many aspects of HD as seen in humans. After preliminary characterization of the human HIP14 BAC transgenic mice, these mice will be crossed to the YAC model of HD. We anticipate that overexpression of HIP14 in vivo will delay and/or ameliorate the features of HD observed in the YAC128 mouse, providing validation of this pathway as a potential therapeutic target for HD. F.B.J.Y. is supported by a Canadian Institutes of Health Research Walter and Jessie Boyd & Charles Scriver and Child & Family Research Institute –UBC MD/PhDStudentship Award. She also receives funding from the Michael Smith Foundation for Health Research as a Junior Trainee.

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