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1.
Cell ; 182(1): 50-58.e8, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32516571

RESUMO

COVID-19 has spread worldwide since 2019 and is now a severe threat to public health. We previously identified the causative agent as a novel SARS-related coronavirus (SARS-CoV-2) that uses human angiotensin-converting enzyme 2 (hACE2) as the entry receptor. Here, we successfully developed a SARS-CoV-2 hACE2 transgenic mouse (HFH4-hACE2 in C3B6 mice) infection model. The infected mice generated typical interstitial pneumonia and pathology that were similar to those of COVID-19 patients. Viral quantification revealed the lungs as the major site of infection, although viral RNA could also be found in the eye, heart, and brain in some mice. Virus identical to SARS-CoV-2 in full-genome sequences was isolated from the infected lung and brain tissues. Last, we showed that pre-exposure to SARS-CoV-2 could protect mice from severe pneumonia. Our results show that the hACE2 mouse would be a valuable tool for testing potential vaccines and therapeutics.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/patologia , Modelos Animais de Doenças , Camundongos Transgênicos , Pneumonia Viral/patologia , Animais , Feminino , Humanos , Doenças Pulmonares Intersticiais/patologia , Doenças Pulmonares Intersticiais/virologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/genética , Pandemias , Peptidil Dipeptidase A/genética , Tropismo Viral , Perda de Peso
2.
PLoS One ; 14(10): e0223052, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31613887

RESUMO

To further investigate the role of the phosphate (Pi) transporter PIT1 in Pi homeostasis and tissue mineralization, we developed a transgenic mouse expressing the C-terminal influenza hemagglutinin (HA) epitope-tagged human PIT1 transporter under control of the cytomegalovirus/chicken beta actin/rabbit beta-globin gene (CAG) promotor and a loxP-stop-loxP (LSL) cassette permitting conditional activation of transgene expression (LSL-HA-hPITtg/+). For an initial characterization of this conditional mouse model, germline excision of the LSL cassette was performed to induce expression of the transgene in all mouse tissues (HA-hPIT1tg/+). Recombination was confirmed using genomic DNA obtained from blood samples of these mice. Furthermore, expression of HA-hPIT1 was found to be at least 10-fold above endogenous mouse Pit1 in total RNA isolated from multiple tissues and from cultured primary calvaria osteoblasts (PCOB) estimated by semi-quantitative RT-PCR. Robust expression of the HA-hPIT1 protein was also observed upon immunoblot analysis in most tissues and permits HA-mediated immunoprecipitation of the transporter. Characterization of the phenotype of HA-hPIT1tg/+ mice at 80 days of age when fed a standard chow (0.7% Pi and 1% calcium) showed elevated plasma Pi, but normal plasma iPTH, iFGF23, serum calcium, BUN, 1,25-dihydroxy vitamin D levels and urine Pi, calcium and protein excretion when compared to WT littermates. Likewise, no change in bone mineral density was observed upon uCT analysis of the distal femur obtained from these mice. In conclusion, heterozygous overexpression of HA-hPIT1 is compatible with life and causes hyperphosphatemia while bone and mineral metabolism of these mice are otherwise normal.


Assuntos
Efeito Fundador , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Camundongos Transgênicos/genética , Fosfatos/metabolismo , Fator de Transcrição Pit-1/genética , Transgenes , Actinas/genética , Actinas/metabolismo , Animais , Transporte Biológico , Densidade Óssea , Calcitriol/sangue , Galinhas , Citomegalovirus/genética , Citomegalovirus/metabolismo , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Hormônio Paratireóideo/genética , Hormônio Paratireóideo/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Crânio/citologia , Crânio/metabolismo , Fator de Transcrição Pit-1/metabolismo , Globinas beta/genética , Globinas beta/metabolismo
3.
PLoS Biol ; 17(8): e3000374, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31393866

RESUMO

A deep understanding of how regulation of the multiple levels of gene expression in mammalian tissues give rise to complex phenotypes has been impeded by cellular diversity. A handful of techniques were developed to tag-select nucleic acids of interest in specific cell types, thereby enabling their capture. We expanded this strategy by developing the Tagger knock-in mouse line bearing a quad-cistronic transgene combining enrichment tools for nuclei, nascent RNA, translating mRNA, and mature microRNA (miRNA). We demonstrate that Tagger can capture the desired nucleic acids, enabling multiple omics approaches to be applied to specific cell types in vivo using a single transgenic mouse line.


Assuntos
Perfilação da Expressão Gênica/métodos , Ácidos Nucleicos/isolamento & purificação , Sequenciamento Completo do Genoma/métodos , Animais , Clonagem Molecular/métodos , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Técnicas de Introdução de Genes , Genômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/genética , MicroRNAs/genética , Proteômica/métodos , RNA Mensageiro/genética , Transcriptoma/genética , Transgenes/genética
4.
Gene ; 714: 143996, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31348980

RESUMO

The uniquely human α7-nAChR gene (CHRFAM7A) is evolved from the fusion of two partially duplicated genes, FAM7 and α7-nAChR gene (CHRNA7), and is inserted on same chromosome 15, 5' end of the CHRNA7 gene. Transcription of CHRFAM7A gene produces a 1256-bp open reading frame encoding dup-α7-nAChR, where a 27-aminoacid residues from FAM7 replaced the 146-aminoacid residues of the N-terminal extracellular ligand binding domain of α7-nAChR. In vitro, dup-α7-nAChR has been shown to form hetero-pentamer with α7-nAChR and dominant-negatively regulates the channel functions of α7-nAChR. However, the contribution of CHRFAM7A gene to the biology of α7-nAChR in the brain in vivo remains largely a matter of conjecture. CHRFAM7A transgenic mouse was created and differentially expressed proteins were profiled from the whole brain using iTRAQ-2D-LC-MS/MS proteomic technology. Proteins with a fold change of ≥1.2 or ≤0.83 and p < 0.05 were considered to be significant. Bioinformatics analysis showed that over-expression of the CHRFAM7A gene significantly modulated the proteins commonly involved in the signaling pathways of α7-nAChR-mediated neuropsychiatric disorders including Parkinson's disease, Alzheimer's disease, Huntington's disease, and alcoholism, suggesting that the CHRFAM7A gene contributes to the pathogenesis of neuropsychiatric disorders mostly likely through fine-tuning the functions of α7-nAChR in the brain.


Assuntos
Camundongos Transgênicos/genética , Receptor Nicotínico de Acetilcolina alfa7/genética , Animais , Encéfalo/metabolismo , Cromatografia Líquida/métodos , Cromossomos Humanos Par 15/genética , Perfilação da Expressão Gênica/métodos , Genes Duplicados/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteômica/métodos , Transdução de Sinais/genética , Espectrometria de Massas em Tandem/métodos
5.
Gene Expr Patterns ; 34: 119064, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31301385

RESUMO

Many aspects of the functional role of the E3 ubiquitin ligase Hectd1 in embryogenesis and in cell biology still remain to be elucidated. In order to contribute to this task we now report the generation of a new transgenic mouse model for Hectd1 using the gene trap strategy. The HECT domain deletion mutant mouse was created by inserting a ß-geo cassette into the Hectd1 locus. Mice homozygous for Hectd1-mutant showed early embryonic lethality with abnormal placental development and defective of neural tube closure resulting in exencephaly. The thickness of the placenta of both Hectd1-mutant homozygous and heterozygous mice was distinctly thinner than that of wildtype mice, the difference being most pronounced in the labyrinth layer of the placenta. We also addressed the temporal and spatial expression profiles of Hectd1 in adult tissues by X-gal staining. Hectd1 expression was detected in specific cell populations of most but not all tissues of the adult organism. Furthermore, the expression of Hectd1 was regulated by insulin and by both heat and hypoxia. Thus, our studies reveal that Hectd1 is indispensable for normal embryogenesis and fetal survival. The generation of this new Hectd1 mutant mouse model provides ample opportunities to study the function of Hectd1 in mammalian cells in detail.


Assuntos
Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Desenvolvimento Embrionário/genética , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos/embriologia , Camundongos Transgênicos/genética , Placenta/metabolismo , Placentação , Gravidez
7.
Neurochem Res ; 44(8): 1999-2006, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31325154

RESUMO

Adult-onset hypothyroidism induces cognitive impairments in learning and memory. Thyroxin (T4) replacement therapy appears to be effective in biochemically restoring euthyroidism, as evidenced by serum T4 and triiodothyronine concentrations within the normal range, although some the patients still exhibit cognitive dysfunctions. Here, we investigated the cognitive functions of propylthiouracil-induced hypothyroid mice in C57BL/6j and 129/Sv strains using the passive avoidance task and the novel object recognition test. Cognitive dysfunctions in hypothyroid mice were found only in the C57BL/6j strain, not in the 129/Sv strain. Further, we found that cholinergic neurons in the basal forebrain increased the membrane potential and input resistance with decreased capacitance, and that they decreased the amplitude and width of action potential in hypothyroid mice in the C57BL/6j strain but not in those in the 129/Sv strain, compared with the controls for each strain. Additionally, the excitability of cholinergic neurons in the basal forebrain was reduced in the hypothyroid mice in the C57BL/6j strain. These results indicated that transgenic mice with the C57BL/6j genetic background are more suitable for revealing the mechanism underlying hypothyroidism-induced cognitive dysfunction, and that the cholinergic basal forebrain may be the appropriate target for treating cognitive dysfunction in adult-onset hypothyroidism.


Assuntos
Disfunção Cognitiva/fisiopatologia , Modelos Animais de Doenças , Hipotireoidismo/fisiopatologia , Camundongos da Linhagem 129/genética , Camundongos Endogâmicos C57BL/genética , Camundongos Transgênicos/genética , Animais , Prosencéfalo Basal/metabolismo , Neurônios Colinérgicos/metabolismo , Disfunção Cognitiva/etiologia , Hipotireoidismo/induzido quimicamente , Hipotireoidismo/complicações , Aprendizagem/fisiologia , Masculino , Potenciais da Membrana/fisiologia , Memória/fisiologia , Propiltiouracila , Hormônios Tireóideos/metabolismo
8.
Mol Carcinog ; 58(9): 1691-1700, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31237025

RESUMO

Non-melanoma skin cancer frequently results from chronic exposure to ultraviolet (UV) irradiation. UV-induced DNA damage activates cell cycle arrest checkpoints through degradation of the cyclin-dependent kinase activators, the cell division cycle 25 (CDC25) phosphatases. We previously reported increased CDC25A in nonmelanoma skin cancer, but CDC25B and CDC25C had not been previously examined. Consequently, we hypothesized that increased expression of CDC25B and CDC25C increases tumor cell proliferation and skin tumor growth. We found that CDC25B and CDC25C were increased in mouse and human skin cancers. CDC25B was primarily cytoplasmic in skin and skin tumors and was significantly increased in the squamous cell carcinoma (SCC), while CDC25C was mostly nuclear in the skin, with an increased cytoplasmic signal in the premalignant and malignant tumors. Surprisingly, forced expression of CDC25B or CDC25C in cultured SCC cells did not affect proliferation, but instead suppressed apoptosis, while CDC25C silencing increased apoptosis without impacting proliferation. Targeting CDC25C to the nucleus via mutation of its nuclear export sequence, however, increased proliferation in SCC cells. Overexpression of CDC25C in the nuclear compartment did not hinder the ability of CDC25C to suppress apoptosis, neither did mutation of sites necessary for its interaction with 14-3-3 proteins. Analysis of apoptotic signaling pathways revealed that CDC25C increased activating phosphorylation of Akt on Ser473 , increased inhibitory phosphorylation of proapoptotic BAD on Ser136 , and increased the survival protein Survivin. Silencing of CDC25C significantly reduced Survivin levels. Taken together, these data suggest that increased expression of CDC25B or CDC25C are mechanisms by which skin cancers evade apoptotic cell death.


Assuntos
Morte Celular/genética , Neoplasias Cutâneas/genética , Fosfatases cdc25/genética , Animais , Apoptose/genética , Carcinoma de Células Escamosas/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Proliferação de Células/genética , Citoplasma/genética , Dano ao DNA/genética , Humanos , Camundongos , Camundongos Transgênicos/genética , Fosforilação/genética , Transdução de Sinais/genética
9.
Dev Dyn ; 248(9): 784-794, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31219647

RESUMO

BACKGROUND: Although Tokudaia muenninki has multiple extra copies of the Sry gene on the Y chromosome, loss of function of these sequences is indicated. To examine the Sry gene function for sex determining in T. muenninki, we screened a BAC library and identified a clone (SRY26) containing complete SRY coding and promoter sequences. RESULTS: SRY26 showed high identity to mouse and rat SRY. In an in vitro reporter gene assay, SRY26 was unable to activate testis-specific enhancer of Sox9. Four lines of BAC transgenic mice carrying SRY26 were generated. Although the embryonic gonads of XX transgenic mice displayed sufficient expression levels of SRY26 mRNA, these mice exhibited normal female phenotypes in the external and internal genitalia, and up-regulation of Sox9 was not observed. Expression of the SRY26 protein was confirmed in primate-derived COS7 cells transfected with a SRY26 expression vector. However, the SRY26 protein was not expressed in the gonads of BAC transgenic mice. CONCLUSIONS: Overall, these results support a previous study demonstrated a long Q-rich domain plays essential roles in protein stabilization in mice. Therefore, the original aim of this study, to examine the function of the Sry gene of this species, was not achieved by creating TG mice.


Assuntos
Genes sry , Proteína da Região Y Determinante do Sexo/genética , Cromossomo Y/genética , Animais , Gônadas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos/genética , Estabilidade Proteica , Ratos , Fatores de Transcrição SOX9/metabolismo , Proteína da Região Y Determinante do Sexo/química , Testículo/metabolismo
10.
Genesis ; 57(9): e23305, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31087513

RESUMO

The mechanisms by which retinal ganglion cells (RGCs) make specific connections during development is an intense area of research and have served as a model for understanding the general principles of circuit wiring. As such, genetic tools allowing for specific recombination in RGCs are critical to further our understanding of the cell-specific roles of different genes during these processes. However, many RGC-specific Cre lines have drawbacks, due to their broad expression in other cell types and/or retinorecipient regions or lack of expression in broad swaths of the retina. Here, we characterize a Cre BAC transgenic line driven by elements of the cholinergic receptor nicotinic beta 3 subunit (Chrnb3). We show that Cre expression is restricted to RGCs in the retina and sparsely expressed in the brain, importantly excluding retinorecipient regions. Furthermore, Chrnb3-Cre mice label a wide variety of RGCs distributed throughout the retina and Cre activity is detected embryonically, shortly following RGC differentiation. Finally, we find that Chrnb3-Cre-labeled RGCs innervate multiple retinorecipient areas that serve both image-forming and nonimage forming functions. Thus, this genetic tool will be of broad use to investigators studying the RGC-specific contributions of genes to visual circuit development.


Assuntos
Cromossomos Artificiais Bacterianos , Regulação da Expressão Gênica , Camundongos Transgênicos/genética , Receptores Nicotínicos/genética , Células Ganglionares da Retina/metabolismo , Animais , Técnicas de Transferência de Genes , Integrases/genética
11.
Genesis ; 57(9): e23304, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31077553

RESUMO

Tissue-specific inducible Cre recombinase mouse lines allow precise genetic manipulations in spatiotemporal manners and are pivotal for functional studies of genes during development and in adults. Growth factor independence 1 (GFI1) is an essential transcription factor expressed in the hair cells of mouse inner ear and Gfi1 locus serves as an excellent anchor site to drive the expression of inducible Cre recombinase in mouse inner hair cells. In this study, we have generated Gfi1-P2A-GFP-CreERT2 (Gfi1-GCE) knock-in mouse line by in-frame fusion of a self-cleaving GCE to the C-terminus of GFI1. We have shown that as predicted, the expression of GCE and GFI1 was detected specifically in the cytosol and nuclei of hair cells, respectively, of uninduced Gfi1-GCE mice, suggesting the successful cleavage and simultaneous expression of GFI1 and GCE. In addition, the in-frame fusion of the self-cleaving GCE does not interrupt the function of Gfi1 in the inner ear. Administration of tamoxifen leads to nuclear translocation of GCE and results in an efficient activation of tdTomato reporter gene expression specifically in most hair cells throughout development and in adults. Thus, this inducible Gfi1-GCE mouse line is a highly efficient Cre deleter and is suitable for gene manipulation in developing and adult inner ear hair cells.


Assuntos
Proteínas de Ligação a DNA/genética , Células Ciliadas Auditivas Internas/metabolismo , Integrases/genética , Camundongos Transgênicos/genética , Fatores de Transcrição/genética , Animais , Técnicas de Introdução de Genes , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/genética , Recombinação Genética
12.
Cells ; 8(4)2019 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-31027317

RESUMO

Inducible cyclization recombinase (Cre) transgenic mouse strains are powerful tools for cell lineage tracing and tissue-specific knockout experiments. However, low efficiency or leaky expression can be important pitfalls. Here, we compared the efficiency and specificity of two commonly used cholangiocyte-specific Cre drivers, the Opn-iCreERT2 and Ck19-CreERT drivers, using a tdTomato reporter strain. We found that Opn-iCreERT2 triggered recombination of the tdTomato reporter in 99.9% of all cholangiocytes while Ck19-CreERT only had 32% recombination efficiency after tamoxifen injection. In the absence of tamoxifen, recombination was also induced in 2% of cholangiocytes for the Opn-iCreERT2 driver and in 13% for the Ck19-CreERT driver. For both drivers, Cre recombination was highly specific for cholangiocytes since recombination was rare in other liver cell types. Toxic liver injury ectopically activated Opn-iCreERT2 but not Ck19-CreERT expression in hepatocytes. However, ectopic recombination in hepatocytes could be avoided by applying a three-day long wash-out period between tamoxifen treatment and toxin injection. Therefore, the Opn-iCreERT2 driver is best suited for the generation of mutant bile ducts, while the Ck19-CreERT driver has near absolute specificity for bile duct cells and is therefore favorable for lineage tracing experiments.


Assuntos
Engenharia Genética/métodos , Queratina-19/metabolismo , Osteopontina/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Ductos Biliares/metabolismo , Linhagem da Célula/efeitos dos fármacos , Feminino , Expressão Gênica/genética , Expressão Gênica/fisiologia , Integrases/biossíntese , Integrases/genética , Integrases/metabolismo , Queratina-19/genética , Queratina-19/fisiologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos/genética , Osteopontina/genética , Osteopontina/fisiologia , Proteínas Recombinantes/metabolismo , Tamoxifeno/farmacologia
13.
J Biotechnol ; 296: 83-92, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-30898688

RESUMO

Silkworms are an economically important insect.Silkworm pupae are also a nutrient-rich food and can be used as a pharmaceutical intermediate.The N-terminus of Aß includes 1-15 amino acid residues with a B cell surface antigen that is necessary to produce antibody and prevent the adverse reactions observed in response to the full Aß42 peptide. In this study, we used silkworm pupae to develop a safer vaccine for Alzheimer's disease (AD) patients. Aß15 peptide was fused with the cholera toxin B subunit (CTB) and expressed in silkworm pupae. Then, we tested an oral vaccine with the peptide expressed by silkworm pupae in a transgenic mouse model of AD. The results show that anti-Aß antibodies were induced, Aß deposition in the brain decreased, the content of malondialdehyde was lower than in the other group, and memory and cognition of the mice improved. These results suggest that the high-nutrient CTB-Aß15 silkworm pupa vaccine has a potential clinical application for the prevention of AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/genética , Toxina da Cólera/administração & dosagem , Vacinas/administração & dosagem , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/imunologia , Animais , Bombyx/química , Bombyx/genética , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Transgênicos/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Pupa/genética , Pupa/crescimento & desenvolvimento , Vacinas/genética , Vacinas/imunologia
14.
Sci Data ; 6: 190028, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30806643

RESUMO

The spatial pattern of transgene expression in tetracycline-controlled mouse models is governed primarily by the driver line used to introduce the tetracycline-controlled transactivator (tTA). Detailed maps showing where each tTA driver activates expression are therefore essential for designing and using tet-regulated models, particularly in brain research where cell type and regional specificity determine the circuits affected by conditional gene expression. We have compiled a comprehensive online repository of serial microscopic images showing brain-wide reporter expression for five commonly used tTA driver lines. We have spatially registered all images to a common three-dimensional mouse brain anatomical reference atlas for direct comparison of spatial distribution across lines. The high-resolution images and associated metadata are shared via the web page of the EU Human Brain Project. Images can be inspected using an interactive viewing tool that includes an optional overlay feature providing anatomical delineations and reference atlas coordinates. Interactive viewing is supplemented by semi-quantitative analyses of expression levels within anatomical subregions for each tTA driver line.


Assuntos
Mapeamento Encefálico/métodos , Regulação da Expressão Gênica , Genes Reporter , Camundongos Transgênicos , Animais , Regulação da Expressão Gênica/fisiologia , Imageamento Tridimensional , Camundongos , Camundongos Transgênicos/anatomia & histologia , Camundongos Transgênicos/genética , Regiões Promotoras Genéticas , Tetraciclina , Transativadores/fisiologia
15.
Proc Natl Acad Sci U S A ; 116(9): 3703-3711, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808757

RESUMO

One of the strongest susceptibility genes for age-related macular degeneration (AMD) is complement factor H (CFH); however, its impact on AMD pathobiology remains unresolved. Here, the effect of the principal AMD-risk-associated CFH variant (Y402H) on the development and progression of age-dependent AMD-like pathologies was determined in vivo. Transgenic mice expressing equal amounts of the full-length normal human CFH Y402 (CFH-Y/0) or the AMD-risk associated CFH H402 (CFH-H/H) variant on a Cfh -/- background were aged to 90 weeks and switched from normal diet (ND) to a high fat, cholesterol-enriched (HFC) diet for 8 weeks. The resulting phenotype was compared with age-matched controls maintained on ND. Remarkably, an AMD-like phenotype consisting of vision loss, increased retinal pigmented epithelium (RPE) stress, and increased basal laminar deposits was detected only in aged CFH-H/H mice following the HFC diet. These changes were not observed in aged CFH-Y/0 mice or in younger (36- to 40-week-old) CFH mice of both genotypes fed either diet. Biochemical analyses of aged CFH mice after HFC diet revealed genotype-dependent changes in plasma and eyecup lipoproteins, but not complement activation, which correlated with the AMD-like phenotype in old CFH-H/H mice. Specifically, apolipoproteins B48 and A1 are elevated in the RPE/choroid of the aged CFH-H/H mice compared with age-matched control CFH-Y/0 fed a HFC diet. Hence, we demonstrate a functional consequence of the Y402H polymorphism in vivo, which promotes AMD-like pathology development and affects lipoprotein levels in aged mice. These findings support targeting lipoproteins as a viable therapeutic strategy for treating AMD.


Assuntos
Ativação do Complemento/genética , Fator H do Complemento/genética , Lipoproteínas/genética , Degeneração Macular/genética , Animais , Dieta Hiperlipídica/efeitos adversos , Feminino , Genótipo , Humanos , Lipoproteínas/metabolismo , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Transgênicos/genética , Polimorfismo de Nucleotídeo Único/genética , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia
16.
Endocrinol Metab (Seoul) ; 34(1): 11-22, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30784243

RESUMO

The development of next generation sequencing (NGS) has led to marked advancement of our understanding of genetic events mediating the initiation and progression of thyroid cancers. The NGS studies have confirmed the previously reported high frequency of mutually-exclusive oncogenic alterations affecting BRAF and RAS proto-oncogenes in all stages of thyroid cancer. Initially identified by traditional sequencing approaches, the NGS studies also confirmed the acquisition of alterations that inactivate tumor protein p53 (TP53) and activate phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) in advanced thyroid cancers. Novel alterations, such as those in telomerase reverse transcriptase (TERT) promoter and mating-type switching/sucrose non-fermenting (SWI/SNF) complex, are also likely to promote progression of the BRAFV600E-driven thyroid cancers. A number of genetically engineered mouse models (GEMM) of BRAFV600E-driven thyroid cancer have been developed to investigate thyroid tumorigenesis mediated by oncogenic BRAF and to explore the role of genetic alterations identified in the genomic analyses of advanced thyroid cancer to promote tumor progression. This review will discuss the various GEMMs that have been developed to investigate oncogenic BRAFV600E-driven thyroid cancers.


Assuntos
Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proto-Oncogenes/genética , Neoplasias da Glândula Tireoide/genética , Animais , Carcinoma Papilar/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Progressão da Doença , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Camundongos Transgênicos/genética , Mutação , Telomerase/genética , Neoplasias da Glândula Tireoide/veterinária , Proteína Supressora de Tumor p53/metabolismo
17.
Genetics ; 211(4): 1155-1177, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30765420

RESUMO

To understand gene function, the cre/loxP conditional system is the most powerful available for temporal and spatial control of expression in mouse. However, the research community requires more cre recombinase expressing transgenic mouse strains (cre-drivers) that restrict expression to specific cell types. To address these problems, a high-throughput method for large-scale production that produces high-quality results is necessary. Further, endogenous promoters need to be chosen that drive cell type specific expression, or we need to further focus the expression by manipulating the promoter. Here we test the suitability of using knock-ins at the docking site 5' of Hprt for rapid development of numerous cre-driver strains focused on expression in adulthood, using an improved cre tamoxifen inducible allele (icre/ERT2), and testing a novel inducible-first, constitutive-ready allele (icre/f3/ERT2/f3). In addition, we test two types of promoters either to capture an endogenous expression pattern (MaxiPromoters), or to restrict expression further using minimal promoter element(s) designed for expression in restricted cell types (MiniPromoters). We provide new cre-driver mouse strains with applicability for brain and eye research. In addition, we demonstrate the feasibility and applicability of using the locus 5' of Hprt for the rapid generation of substantial numbers of cre-driver strains. We also provide a new inducible-first constitutive-ready allele to further speed cre-driver generation. Finally, all these strains are available to the research community through The Jackson Laboratory.


Assuntos
Encéfalo/metabolismo , Olho/metabolismo , Técnicas de Introdução de Genes/métodos , Camundongos Transgênicos/genética , Tamoxifeno/farmacologia , Ativação Transcricional/efeitos dos fármacos , Animais , Efeito Fundador , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas
18.
FASEB J ; 33(4): 5571-5584, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30640520

RESUMO

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology facilitates somatic genome editing to reveal cooperative genetic interactions at the cellular level without extensive breeding between different mutant animals. Here we propose a transgenic inducible Cas9 effector-CRISPR mutagen ( ICE CRIM) mouse model in which CRISPR/Cas9-mediated somatic mutagenesis events can occur in response to Cre expression. The well-known tumor suppressor gene, Trp53, and 2 important DNA mismatch repair genes, Mlh1 and Msh2, were selected to be our somatic mutagenesis targets. Amplicon-based sequencing was performed to validate the editing efficiency and to identify the mutant allelic series. Crossed with various Cre lines, the Trp53 ICE CRIM alleles were activated to generate targeted cancer gene somatic or germ line mutant variants. We provide experimental evidence to show that an activated ICE CRIM can mutate both targeted alleles within a cell. Simultaneous disruption of multiple genes was also achieved when there were multiple single-guide RNA expression cassettes embedded within an activated ICE CRIM. Our mouse model can be used to generate mutant pools in vivo, which enables a functional screen to be performed in situ. Our results also provide evidence to support a monoclonal origin of hematopoietic neoplasms and to indicate that DNA mismatch repair deficiency accelerates tumorigenesis in Trp53 mutant genetic background.-Fan, H.-H., Yu, I.-S., Lin, Y.-H., Wang, S.-Y., Liaw, Y.-H., Chen, P.-L., Yang, T.-L., Lin, S.-W., Chen, Y.-T. P53 ICE CRIM mouse: a tool to generate mutant allelic series in somatic cells and germ lines for cancer studies.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Mutação/genética , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Alelos , Animais , Edição de Genes/métodos , Marcação de Genes/métodos , Células Germinativas , Camundongos , Camundongos Transgênicos/genética , Mutagênese/genética , Oncogenes/genética , RNA Guia/genética
19.
G3 (Bethesda) ; 9(2): 591-599, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30591434

RESUMO

The modification of the mouse genome by site-specific gene insertion of transgenes and other genetic elements allows the study of gene function in different developmental stages and in the pathogenesis of diseases. Here, we generated a "genomic safe harbor" Hipp11 (H11) locus-specific knock-in transgenic mouse line in which the albumin promoter is used to drive the expression of the reverse tetracycline transactivator (rtTA) in the liver. The newly generated H11-albumin-rtTA transgenic mice were bred with tetracycline-operator-Histone-2B-green fluorescent protein (TetO-H2BGFP) mice to assess inducibility and tissue-specificity. Expression of the H2BGFP fusion protein was observed exclusively upon doxycycline (Dox) induction in the liver of H11-albumin-rtTA/TetO-H2BGFP double transgenic mice. To further analyze the ability of the Dox-inducible H11-albumin-rtTA mice to implement conditional DNA recombination, H11-albumin-rtTA transgenic mice were crossed with TetO-Cre and Ai14 mice to generate H11-albumin-rtTA/TetO-Cre/Ai14 triple transgenic mice. We successfully confirmed that the Cre-mediated recombination efficiency was as strong in Dox-induced H11-albumin-rtTA /TetO-Cre/Ai14 mice as in the control albumin-Cre/A14 mice. Finally, to characterize the expression-inducing effects of Dox in H11-albumin-rtTA/TetO-H2BGFP mice in detail, we examined GFP expression in embryos at different developmental stages and found that newly conceived H11-albumin-rtTA/TetO-H2BGFP embryos of Dox-treated pregnant female mice were expressing reporter GFP by E16.5. Our study demonstrates that these new H11-albumin-rtTA transgenic mice are a powerful and efficient tool for the temporally and spatially conditional manipulation of gene expression in the liver, and illustrates how genetic crosses with these new mice enable the generation of complex multi-locus transgenic animals for mechanistic studies.


Assuntos
Técnicas de Introdução de Genes/métodos , Fígado/metabolismo , Camundongos Transgênicos/genética , Albuminas/genética , Albuminas/metabolismo , Animais , Doxiciclina/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Regiões Promotoras Genéticas , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional/efeitos dos fármacos
20.
J Cell Mol Med ; 23(1): 568-575, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30334333

RESUMO

Histone lysine methylation plays an important role in the regulation of ventricular remodelling. NSD2 is involved in many types of tumours through enhancing H3K36me2 expression. However, the role of NSD2 in the regulation of histone lysine methylation during ventricular remodelling remains unclear. In this study, we established cardiac hypertrophy model in C57BL/6 mice by transverse aortic constriction and found that histone lysine methylation participated in ventricular remodelling regulation via the up-regulation of H3K27me2 and H3K36me2 expression. In addition, we constructed transgenic C57BL/6 mice with conditional knockout of NSD2 (NSD2-/- ) in the myocardium. NSD2-/- C57BL/6 mice had milder ventricular remodelling and significantly improved cardiac function compared with wild-type mice, and the expression of H3K36me2 but not H3K27me2 was down-regulated. In conclusion, NSD2 promotes ventricular remodelling mediated by the regulation of H3K36me2.


Assuntos
Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Remodelação Ventricular/genética , Animais , Regulação para Baixo/genética , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/genética , Miocárdio , Regulação para Cima/genética
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