RESUMO
Non-alcoholic fatty liver disease (NAFLD) constitutes a metabolic disorder with high worldwide prevalence and increasing incidence. The inflammatory progressive state, non-alcoholic steatohepatitis (NASH), leads to liver fibrosis and carcinogenesis. Here, we evaluated whether tyrosinase mutation underlies NASH pathophysiology. Tyrosinase point-mutated B6 (Cg)-Tyrc-2J/J mice (B6 albino) and C57BL/6J black mice (B6 black) were fed with high cholesterol diet (HCD) for 10 weeks. Normal diet-fed mice served as controls. HCD-fed B6 albino exhibited high NASH susceptibility compared to B6 black, a phenotype not previously reported. Liver injury occurred in approximately 50% of B6 albino from one post HCD feeding, with elevated serum alanine aminotransferase and aspartate aminotransferase levels. NASH was induced following 2 weeks in severe-phenotypic B6 albino (sB6), but B6 black exhibited no symptoms, even after 10 weeks. HCD-fed sB6 albino showed significantly higher mortality rate. Histological analysis of the liver revealed significant inflammatory cell and lipid infiltration and severe fibrosis. Serum lipoprotein analysis revealed significantly higher chylomicron and very low-density lipoprotein levels in sB6 albino. Moreover, significantly higher small intestinal lipid absorption and lower fecal lipid excretion occurred together with elevated intestinal NPC1L1 expression. As the tyrosinase point mutation represents the only genetic difference between B6 albino and B6 black, our work will facilitate the identification of susceptible genetic factors for NASH development and expand the understanding of NASH pathophysiology.
Assuntos
Colesterol na Dieta/administração & dosagem , Colesterol na Dieta/efeitos adversos , Monofenol Mono-Oxigenase/genética , Hepatopatia Gordurosa não Alcoólica/etiologia , Mutação Puntual , Albinismo Oculocutâneo/complicações , Albinismo Oculocutâneo/enzimologia , Albinismo Oculocutâneo/genética , Animais , Colesterol/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Humanos , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Lipoproteínas/sangue , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/genéticaRESUMO
Sendai virus (SeV) vectors are being recognized as a superior tool for gene transfer. Here, we report the transfection efficacy of a novel, high-performance, replication-defective, and persistent Sendai virus (SeVdp) vector in cultured cells and in mice using a near-infrared fluorescent protein (iRFP)-mediated in vivo imaging system. The novel SeVdp vector established persistent infection, and strong expression of inserted genes was sustained indefinitely in vitro. Analysis of iRFP-expressing cells transplanted subcutaneously into NOG, nude, and ICR mice suggests that innate immunity was involved in the exclusion of the transplanted cells. We also evaluated the feasibility of this novel SeVdp vector for hemophilia A gene therapy. This system enabled insertion of full-length FVIII genes, and transduced cells secreted FVIII into the culture medium. Transient FVIII activity was detected in the plasma of mice after intraperitoneal transplantation of these FVIII-secreting cells. Further improvement in methods to evade immunity, such as simultaneous expression of immunomodulatory genes, would make this novel vector a very useful tool in regenerative medicine.
Assuntos
Terapia Genética , Vetores Genéticos/genética , Hemofilia A/genética , Hemofilia A/terapia , Vírus Sendai/genética , Animais , Testes de Coagulação Sanguínea , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Modelos Animais de Doenças , Fator VIII/genética , Expressão Gênica , Ordem dos Genes , Técnicas de Transferência de Genes , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Humanos , Camundongos , Camundongos Knockout , Transdução Genética , TransgenesRESUMO
By using near-infrared fluorescent protein (iRFP)-expressing hematopoietic cells, we established a novel, quantitative, in vivo, noninvasive atherosclerosis imaging system. This murine atherosclerosis imaging approach targets macrophages expressing iRFP in plaques. Low-density lipoprotein receptor-deficient (LDLR-/-) mice transplanted with beta-actin promoter-derived iRFP transgenic (TG) mouse bone marrow (BM) cells (iRFP â LDLR-/-) were used. Atherosclerosis was induced by a nonfluorescent 1.25% cholesterol diet (HCD). Atherosclerosis was compared among the three differently induced mouse groups. iRFP â LDLR-/- mice fed a normal diet (ND) and LDLR-/- mice transplanted with wild-type (WT) BM cells were used as controls. The in vivo imaging system (IVIS) detected an enhanced iRFP signal in the thoracic aorta of HCD-fed iRFP â LDLR-/- mice, whereas iRFP signals were not observed in the control mice. Time-course imaging showed a gradual increase in the signal area, which was correlated with atherosclerotic plaque progression. Oil red O (ORO) staining of aortas and histological analysis of plaques confirmed that the detected signal was strictly emitted from plaque-positive areas of the aorta. Our new murine atherosclerosis imaging system can noninvasively image atherosclerotic plaques in the aorta and generate longitudinal data, validating the ability of the system to monitor lesion progression.
Assuntos
Doenças da Aorta/diagnóstico por imagem , Aterosclerose/diagnóstico por imagem , Medições Luminescentes/métodos , Proteínas Luminescentes/análise , Imagem Óptica/métodos , Placa Aterosclerótica/diagnóstico por imagem , Actinas/genética , Animais , Doenças da Aorta/genética , Aterosclerose/etiologia , Aterosclerose/genética , Compostos Azo , Transplante de Medula Óssea , Colesterol na Dieta/toxicidade , Corantes , Citometria de Fluxo , Genes Reporter , Genes Sintéticos , Proteínas Luminescentes/genética , Macrófagos Peritoneais/química , Macrófagos Peritoneais/ultraestrutura , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Receptores de LDL/deficiência , Proteínas Recombinantes/análise , Proteínas Recombinantes/genéticaRESUMO
The in vivo imaging of mice makes it possible to analyze disease progress non-invasively through reporter gene expression. As the removal of hair improves the accuracy of in vivo imaging, gene-modified mice with a reporter gene are often crossed with Hos:HR-1 mutant mice homozygous for the spontaneous Hrhr mutation that exhibit a hair loss phenotype. However, it is time consuming to produce mice carrying both the reporter gene and mutant Hrhr gene by mating. In addition, there is a risk that genetic background of the gene-modified mice would be altered by mating. To resolve these issues, we established a simple method to generate hairless mice maintaining the original genetic background by CRISPR technology. First, we constructed the pX330 vector, which targets exon 3 of Hr. This DNA vector (5 ng/µl) was microinjected into the pronuclei of C57BL/6J mice. Induced Hr gene mutations were found in many founders (76.1%) and these mutations were heritable. Next, we performed in vivo imaging using these gene-modified hairless mice. As expected, luminescent objects in their body were detected by in vivo imaging. This study clearly showed that hairless mice could be simply generated by the CRISPR/Cas9 system, and this method may be useful for in vivo imaging studies with various gene-modified mice.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Diagnóstico por Imagem/métodos , Camundongos Pelados/genética , Terapia de Substituição Mitocondrial/métodos , Mutação , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , DNA/genética , Genes Reporter/genética , Vetores Genéticos , Camundongos Endogâmicos C57BL , Microinjeções , FenótipoRESUMO
Angiogenesis is important for normal development as well as for tumour growth. However, the molecular and cellular mechanisms underlying angiogenesis are not fully understood, partly because of the lack of a good animal model for imaging. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a bioluminescent reporter protein, Nano-lantern, under the control of Fetal liver kinase 1 (Flk1). Flk1-Nano-lantern BAC Tg mice recapitulated endogenous Flk1 expression in endothelial cells and lymphatic endothelial cells during development and tumour growth. Importantly, bioluminescence imaging of endothelial cells from the aortic rings of Flk1-Nano-lantern BAC Tg mice enabled us to observe endothelial sprouting for 18 hr without any detectable phototoxicity. Furthermore, Flk1-Nano-lantern BAC Tg mice achieved time-lapse luminescence imaging of tumour angiogenesis in freely moving mice with implanted tumours. Thus, this transgenic mouse line contributes a unique model to study angiogenesis within both physiological and pathological contexts.