RESUMO
WIZ (Widely Interspaced Zinc Finger) is associated with the G9a-GLP protein complex, a key H3K9 methyltransferase suggesting a role in transcriptional repression. However, its role in embryonic development is poorly described. In order to assess the loss of function of WIZ, we generated CRISPR/Cas9 WIZ knockout mouse model with 32 nucleotide deletion. Observing the lethality status, we identified the WIZ knockouts to be subviable during embryonic development and non-viable after birth. Morphology of developing embryo was analyzed at E14.5 and E18.5 and our findings were supported by microCT scans. Wiz KO showed improper development in multiple aspects, specifically in the craniofacial area. In particular, shorter snout, cleft palate, and cleft eyelids were present in mutant embryos. Palatal shelves were hypomorphic and though elevated to a horizontal position on top of the tongue, they failed to make contact and fuse. By comparison of proliferation pattern and histone methylation in developing palatal shelves we brought new evidence of importance WIZ dependent G9a-GLP methylation complex in craniofacial development, especially in palate shelf fusion.
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Reporter gene mouse lines are routinely used for studies related to functional genomics, proteomics, cell biology or cell-based drug screenings, and represent a crucial platform for in vivo research. In the generation of knock-in reporter lines, new gene targeting methods provide several advantages over the standard transgenic techniques. First of all, specific targeting of the genome allows expression of the reporter gene under controlled conditions, whether in a specific locus in the genome or in a "safe harbor" locus. Historically, the ROSA26 locus is used for gene knock-in strategies by homologous recombination in mouse embryonic stem cells. The other preferred place for integration of the reporter transgene in the mouse genome is the endogenous promoter of a target gene. In this study, we employed TALENs to generate a reporter fusion protein expressed from its native promoter. For monitoring DNA damage response, we generated a mouse line expressing a mCitrine-tagged version of the FANCD2 protein, involved in DNA damage response and repair, and the Fanconi anemia (FA) pathway. This model could be a valuable tool for in vivo investigation of DNA damage.
Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Genes Reporter , Modelos Animais , Proteínas Recombinantes de Fusão , Animais , Proteínas de Bactérias , Dano ao DNA , Reparo do DNA , Proteínas Luminescentes , Camundongos , Camundongos Endogâmicos C57BL , Nucleases dos Efetores Semelhantes a Ativadores de TranscriçãoRESUMO
Zinc finger 644 (Zfp644 in mouse, ZNF644 in human) gene is a transcription factor whose mutation S672G is considered a potential genetic factor of inherited high myopia. ZNF644 interacts with G9a/GLP complex, which functions as a H3K9 methyltransferase to silence transcription. In this study, we generated mouse models to unravel the mechanisms leading to symptoms associated with high myopia. Employing TALEN technology, two mice mutants were generated, either with the disease-carrying mutation (Zfp644 S673G ) or with a truncated form of Zfp644 (Zfp644 Δ8 ). Eye morphology and visual functions were analysed in both mutants, revealing a significant difference in a vitreous chamber depth and lens diameter, however the physiological function of retina was preserved as found under the high-myopia conditions. Our findings prove that ZNF644/Zfp644 is involved in the development of high-myopia, indicating that mutations such as, Zfp644 S673G and Zfp644 Δ8 are causative for changes connected with the disease. The developed models represent a valuable tool to investigate the molecular basis of myopia pathogenesis and its potential treatment.
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Netherton syndrome (NS) is a severe skin disease caused by the loss of protease inhibitor LEKTI, which leads to the dysregulation of epidermal proteases and severe skin-barrier defects. KLK5 was proposed as a major protease in NS pathology, however its inactivation is not sufficient to rescue the lethal phenotype of LEKTI-deficient mice. In this study, we further elucidated the in vivo roles of the epidermal proteases in NS using a set of mouse models individually or simultaneously deficient for KLK5 and KLK7 on the genetic background of a novel NS-mouse model. We show that although the ablation of KLK5 or KLK7 is not sufficient to rescue the lethal effect of LEKTI-deficiency simultaneous deficiency of both KLKs completely rescues the epidermal barrier and the postnatal lethality allowing mice to reach adulthood with fully functional skin and normal hair growth. We report that not only KLK5 but also KLK7 plays an important role in the inflammation and defective differentiation in NS and KLK7 activity is not solely dependent on activation by KLK5. Altogether, these findings show that unregulated activities of KLK5 and KLK7 are responsible for NS development and both proteases should become targets for NS therapy.
Assuntos
Calicreínas/genética , Síndrome de Netherton/genética , Fenótipo , Animais , Deleção de Genes , Camundongos , Síndrome de Netherton/patologia , Inibidor de Serinopeptidase do Tipo Kazal 5 , Serpinas/genéticaRESUMO
TMEM70, a 21-kDa protein localized in the inner mitochondrial membrane, has been shown to facilitate the biogenesis of mammalian F1Fo ATP synthase. Mutations of the TMEM70 gene represent the most frequent cause of isolated ATP synthase deficiency resulting in a severe mitochondrial disease presenting as neonatal encephalo-cardiomyopathy (OMIM 604273). To better understand the biological role of this factor, we generated Tmem70-deficient mice and found that the homozygous Tmem70-/- knockouts exhibited profound growth retardation and embryonic lethality at â¼9.5 days post coitum. Blue-Native electrophoresis demonstrated an isolated deficiency in fully assembled ATP synthase in the Tmem70-/- embryos (80% decrease) and a marked accumulation of F1 complexes indicative of impairment in ATP synthase biogenesis that was stalled at the early stage, following the formation of F1 oligomer. Consequently, a decrease in ADP-stimulated State 3 respiration, respiratory control ratio and ATP/ADP ratios, indicated compromised mitochondrial ATP production. Tmem70-/- embryos exhibited delayed development of the cardiovascular system and a disturbed heart mitochondrial ultrastructure, with concentric or irregular cristae structures. Tmem70+/- heterozygous mice were fully viable and displayed normal postnatal growth and development of the mitochondrial oxidative phosphorylation system. Nevertheless, they presented with mild deterioration of heart function. Our results demonstrated that Tmem70 knockout in the mouse results in embryonic lethality due to the lack of ATP synthase and impairment of mitochondrial energy provision. This is analogous to TMEM70 dysfunction in humans and verifies the crucial role of this factor in the biosynthesis and assembly of mammalian ATP synthase.
Assuntos
Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Trifosfato de Adenosina/metabolismo , Animais , Cardiomiopatias/metabolismo , Feminino , Homozigoto , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Erros Inatos do Metabolismo/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/biossíntese , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mutação , Fosforilação Oxidativa , GravidezRESUMO
The 12th Transgenic Technology meeting was held in Edinburgh on 6th-8th October 2014 and interest to participate in the meeting overcame all expectations. The TT2014 was the largest meeting ever with more than 540 scientists, technicians, and students from all over the world. The meeting had an excellent scientific program that brought information on the latest ground-breaking technologies for gene targeting and genome editing using programmable nucleases into the foreground. These presentations were well balanced with several highlights over viewing topics in embryonic stem cell research, embryogenesis, disease models, and animals in agriculture. Ample space was reserved also for short talks presenting technical development and for highlighting posters contributions. A highlight of the meeting was the award of the 10th International Society of Transgenic Technologies Prize to Janet Rossant for her outstanding contributions in the field of mouse embryogenesis.
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Agricultura , Modelos Animais de Doenças , Desenvolvimento Embrionário , Pesquisa com Células-Tronco , Animais , Humanos , CamundongosRESUMO
The epidermis is a stratified tissue composed of different keratinocyte layers that create a barrier protecting the body from external influences, pathogens, and dehydration. The barrier function is mainly achieved by its outermost layer, the stratum corneum. To create a mouse model to study pathophysiological processes in the outermost layers of the epidermis in vivo and in vitro we prepared a construct containing red fluorescent td-Tomato reporter sequence under the control of involucrin promoter and its first intron. Transgenic mice were generated by pronuclear injection and the expression and regulation of the transgene was determined by in vivo imaging and fluorescent microscopy. The promoter targeted the transgene efficiently and specifically into the outermost epidermal layers although weak expression was also found in epithelia of tongue and bladder. The regulation of expression in the epidermis, i.e. fluorescence intensity of the reporter, could be easily followed during wound healing and dermatitis. Thus, these transgenic mice carrying the tdTomato reporter could be used as a valuable tool to study impact of various genes dysregulating the epidermal barrier and to follow effects of therapeutic agents for treatment of skin diseases in vivo.
Assuntos
Diferenciação Celular , Marcação de Genes/métodos , Regiões Promotoras Genéticas , Precursores de Proteínas/genética , Solanum lycopersicum/genética , Cicatrização , Animais , Dermatite/metabolismo , Dermatite/fisiopatologia , Epiderme/metabolismo , Epiderme/fisiopatologia , Epitélio/metabolismo , Epitélio/fisiopatologia , Regulação da Expressão Gênica , Genes Reporter , Imuno-Histoquímica , Íntrons , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Língua/metabolismo , Língua/fisiopatologia , Transgenes , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: Angiogenesis is the process of forming new blood vessels from existing ones and requires degradation of the vascular basement membrane and remodeling of extracellular matrix (ECM) in order to allow endothelial cells to migrate and invade into the surrounding tissue. Matrix metalloproteinases (MMPs) are considered to play a central role in the remodeling of basement membranes and ECM. However, MMPs contribute to vascular remodeling not only by degrading ECM components. Specific MMPs enhance angiogenesis via several ways; they help pericytes to detach from vessels undergoing angiogenesis, release ECM-bound angiogenic growth factors, expose cryptic pro-angiogenic integrin binding sites in the ECM, generate promigratory ECM component fragments, and cleave endothelial cell-cell adhesions. MMPs can also negatively influence the angiogenic process through generating endogenous angiogenesis inhibitors by proteolytic cleavage. Angiostatin, a proteolytic fragment of plasminogen, is one of the most potent antagonists of angiogenesis that inhibits migration and proliferation of endothelial cells. Reports have shown that metalloelastase, pancreas elastase, plasmin reductase, and plasmin convert plasminogen to angiostatin. RESULTS: We report here that MMP-19 processes human plasminogen in a characteristic cleavage pattern to generate three angiostatin-like fragments with a molecular weight of 35, 38, and 42 kDa. These fragments released by MMP-19 significantly inhibited the proliferation of HMEC cells by 27% (p = 0.01) and reduced formation of capillary-like structures by 45% (p = 0.05) compared with control cells. As it is known that angiostatin blocks hepatocyte growth factor (HGF)-induced pro-angiogenic signaling in endothelial cells due to structural similarities to HGF, we have analyzed if the plasminogen fragments generated by MMP-19 interfere with this pathway. As it involves the activation of c-met, the receptor of HGF, we could show that MMP-19-dependent processing of plasminogen decreases the phosphorylation of c-met. CONCLUSION: Altogether, MMP-19 exhibits an anti-angiogenic effect on endothelial cells via generation of angiostatin-like fragments.
Assuntos
Angiostatinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Metaloproteinases da Matriz Secretadas/metabolismo , Fragmentos de Peptídeos/farmacologia , Plasminogênio/metabolismo , Inibidores da Angiogênese/metabolismo , Inibidores da Angiogênese/farmacologia , Capilares/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Metaloproteinases da Matriz Secretadas/genética , Peso Molecular , Fragmentos de Peptídeos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
During the progression of cutaneous melanomas, matrix metalloproteinases (MMPs) facilitate the tumour cells to traverse the basement membrane and invade the dermis. In this study, we analysed the expression of MMP19 in the course of melanoma progression. Although MMP19 was absent in melanocytes and melanoma cells of early stages of melanoma development, its expression was strongly upregulated in the neighbouring keratinocytes that may facilitate the vertical outgrowth of melanoma cells. In contrast to early stages, MMP19 was upregulated during the vertical growth phase of melanoma and in metastases. The upregulation of MMP19 in melanoma of Clark levels IV and V correlates with that of MMP2 and also simultaneously with ceased expression of E-cadherin. To reveal whether MMP19 facilitates the invasion of melanomas, we examined adhesion and migratory capacity of selected melanoma cell lines. Melanoma cell lines with low expression of MMP19 exhibited increased adhesion to various substrates and lower migration in comparison with the cell line with higher expression of MMP19. Moreover, ectopic expression of MMP19 could restore the migratory capacity of melanoma cells with low endogenous level of MMP19. These results suggest that the increase of MMP19 expression hallmarks the progression of cutaneous melanoma and might augment melanoma growth by promoting the invasion of tumour cells.
Assuntos
Metaloproteinases da Matriz Secretadas/biossíntese , Melanoma/enzimologia , Melanoma/patologia , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologia , Western Blotting , Adesão Celular , Movimento Celular , Progressão da Doença , Humanos , Imuno-Histoquímica , Queratinócitos/enzimologia , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , Regulação para CimaRESUMO
Matrix metalloproteinase-19 (MMP19) affects cell proliferation, adhesion, and migration in vitro but its physiological role in vivo is poorly understood. To determine the function of MMP19, we generated mice deficient for MMP19 by disrupting the catalytic domain of mmp19 gene. Although MMP19-deficient mice do not show overt developmental and morphological abnormalities they display a distinct physiological phenotype. In a model of contact hypersensitivity (CHS) MMP19-deficient mice showed impaired T cell-mediated immune reaction that was characterized by limited influx of inflammatory cells, low proliferation of keratinocytes, and reduced number of activated CD8(+) T cells in draining lymph nodes. In the inflamed tissue, the low number of CD8(+) T cells in MMP19-deficient mice correlated with low amounts of proinflammatory cytokines, especially lymphotactin and interferon-inducible T cell alpha chemoattractant (I-TAC). Further analyses showed that T cell populations in the blood of immature, unsensitized mice were diminished and that this alteration originated from an altered maturation of thymocytes. In the thymus, thymocytes exhibited low proliferation rates and the number of CD4(+)CD8(+) double-positive cells was remarkably augmented. Based on the phenotype of MMP19-deficient mice we propose that MMP19 is an important factor in cutaneous immune responses and influences the development of T cells.
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Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Metaloproteinases da Matriz Secretadas/fisiologia , Pele/imunologia , Animais , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Citocinas/biossíntese , Primers do DNA , Citometria de Fluxo , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Ativação Linfocitária , Metaloproteinases da Matriz Secretadas/genética , Camundongos , Camundongos Knockout , Reação em Cadeia da PolimeraseRESUMO
Matrix metalloproteinase-19 (MMP-19), unlike other members of the MMP family, is expressed in basal keratinocytes of intact epidermis whereas keratinocytes in suprabasal and higher epidermal layers express this enzyme only during cutaneous disorders. As the activity of MMP-19 effects proliferation, migration, and adhesion of keratinocytes we examined whether transcription factors involved in keratinocyte differentiation repress the expression of MMP-19. Using luciferase reporter assays, POU transcription factors Tst-1 (Oct-6) and Skn-1a (Oct-11) markedly downregulated the activity of MMP-19 promoter in COS-7 cells and HaCaT keratinocytes. Tst-1 alone was able to inhibit 85% of the promoter activity. Skn-1a exhibited a weak inhibitory effect although it synergistically increased effects of Tst-1. HaCaT cells stably transfected with Tst-1 showed a strong decrease of activity of MMP-19 promoter that correlated with suppression of MMP-19, cytokeratin 14 and 5, decreased cell proliferation, and altered expression of involucrin and loricrin. The expression of MMP-9 was also significantly reduced in Tst-1 expressing keratinocytes. MMP-2 was substantially affected during its activation whereas the expression of MMP-28 was unchanged. Our results suggest that Tst-1 and Skn-1a regulate expression of MMPs in keratinocytes and effect both the expression and activation of these proteolytic enzymes.