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1.
Nat Commun ; 15(1): 5613, 2024 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-38965236

RESUMO

Advancements in CRISPR technology, particularly the development of base editors, revolutionize genetic variant research. When combined with model organisms like zebrafish, base editors significantly accelerate and refine in vivo analysis of genetic variations. However, base editors are restricted by protospacer adjacent motif (PAM) sequences and specific editing windows, hindering their applicability to a broad spectrum of genetic variants. Additionally, base editors can introduce unintended mutations and often exhibit reduced efficiency in living organisms compared to cultured cell lines. Here, we engineer a suite of adenine base editors (ABEs) called ABE-Ultramax (Umax), demonstrating high editing efficiency and low rates of insertions and deletions (indels) in zebrafish. The ABE-Umax suite of editors includes ABEs with shifted, narrowed, or broadened editing windows, reduced bystander mutation frequency, and highly flexible PAM sequence requirements. These advancements have the potential to address previous challenges in disease modeling and advance gene therapy applications.


Assuntos
Adenina , Sistemas CRISPR-Cas , Edição de Genes , Mutação INDEL , Peixe-Zebra , Peixe-Zebra/genética , Animais , Edição de Genes/métodos , Adenina/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Animais Geneticamente Modificados , Alelos
2.
Nat Commun ; 15(1): 5547, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956047

RESUMO

The meninges are critical for the brain functions, but the diversity of meningeal cell types and intercellular interactions have yet to be thoroughly examined. Here we identify a population of meningeal lymphatic supporting cells (mLSCs) in the zebrafish leptomeninges, which are specifically labeled by ependymin. Morphologically, mLSCs form membranous structures that enwrap the majority of leptomeningeal blood vessels and all the mural lymphatic endothelial cells (muLECs). Based on its unique cellular morphologies and transcriptional profile, mLSC is characterized as a unique cell type different from all the currently known meningeal cell types. Because of the formation of supportive structures and production of pro-lymphangiogenic factors, mLSCs not only promote muLEC development and maintain the dispersed distributions of muLECs in the leptomeninges, but also are required for muLEC regeneration after ablation. This study characterizes a newly identified cell type in leptomeninges, mLSC, which is required for muLEC development, maintenance, and regeneration.


Assuntos
Células Endoteliais , Meninges , Peixe-Zebra , Animais , Meninges/citologia , Meninges/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Vasos Linfáticos/citologia , Vasos Linfáticos/metabolismo , Animais Geneticamente Modificados , Linfangiogênese/fisiologia , Regeneração/fisiologia
3.
Xenotransplantation ; 31(1): e12841, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38864375

RESUMO

INTRODUCTION: Orthotopic cardiac xenotransplantation has seen notable improvement, leading to the first compassionate use in 2022. However, it remains challenging to define the clinical application of cardiac xenotransplantation, including the back-up strategy in case of xenograft failure. In this regard, the heterotopic thoracic technique could be an alternative to the orthotopic procedure. We present hemodynamic data of heterotopic thoracic pig-to-baboon transplantation experiments, focusing on perioperative xenograft dysfunction and xenograft overgrowth. METHODS: We used 17 genetically modified piglets as donors for heterotopic thoracic xenogeneic cardiac transplantation into captive-bred baboons. In all animals, pressure probes were implanted in the graft's left ventricle and the recipient's ascending aorta and hemodynamic data (graft pressure, aortic pressure and recipient's heart rate) were recorded continuously. RESULTS: Aortic pressures and heart rates of the recipients' hearts were postoperatively stable in all experiments. After reperfusion, three grafts presented with low left ventricular pressure indicating perioperative cardiac dysfunction (PCXD). These animals recovered from PCXD within 48 h under support of the recipient's heart and there was no difference in survival compared to the other 14 ones. After 48 h, graft pressure increased up to 200 mmHg in all 17 animals with two different time-patterns. This led to a progressive gradient between graft and aortic pressure. With increasing gradient, the grafts stopped contributing to cardiac output. Grafts showed a marked weight increase from implantation to explantation. CONCLUSION: The heterotopic thoracic cardiac xenotransplantation technique is a possible method to overcome PCXD in early clinical trials and an experimental tool to get a better understanding of PCXD. The peculiar hemodynamic situation of increasing graft pressure but missing graft's output indicates outflow tract obstruction due to cardiac overgrowth. The heterotopic thoracic technique should be successful when using current strategies of immunosuppression, organ preservation and donor pigs with smaller body and organ size.


Assuntos
Transplante de Coração , Hemodinâmica , Xenoenxertos , Papio , Transplante Heterólogo , Animais , Transplante Heterólogo/métodos , Transplante de Coração/métodos , Suínos , Hemodinâmica/fisiologia , Sobrevivência de Enxerto , Transplante Heterotópico/métodos , Animais Geneticamente Modificados , Rejeição de Enxerto , Humanos
4.
FASEB J ; 38(13): e23727, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38877845

RESUMO

Oxidative stress is proposed as a regulatory element in various neurological disorders, which is involved in the progress of several neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Antioxidant drugs are widely used to alleviate neurodegenerative disorders. Astragalus membranaceus (Huangqi, AM) is a commonly used medicinal herb with a wide range of pharmacological effects. Here, the protective effect and mechanism of AM extract (AME) and its bioactive compounds against neurodegenerative disorders via alleviating oxidative stress were detected using adult Drosophila melanogaster. The drug safety was measured by development analysis; oxidative stress resistance ability was detected by survival rate under H2O2 environment; ROS level was detected by DHE staining and gstD1-GFP fluoresence assay; antioxidative abilitiy was represent by measuring antioxidant enzyme activity, antioxidative-related gene expression, and ATP and MFN2 levels. The neuroprotective effect was evaluated by lifespan and locomotion analysis in Aß42 transgenic and Pink1B9 mutants. AME dramatically increased the survival rates, improved the CAT activity, restored the decreased mRNA expressions of Sod1, Cat, and CncC under H2O2 stimulation, and ameliorated the neurobehavioral defects of the AD and PD. Thirteen small molecules in AM had antioxidant function, in which vanillic acid and daidzein had the most potent antioxidant effect. Vanillic acid and daidzein could increase the activities of SOD and CAT, GSH level, and the expressions of antioxidant genes. Vanillic acid could improve the levels of ATP and MFN2, and mRNA expressions of ND42 and SDHC to rescue mitochondrial dysfunction. Furthermore, vanillic acid ameliorated neurobehavioral defects of PD. Daidzein ameliorated neurobehavioral defect of Aß-induced AD mode. Taken together, AM plays a protective role in oxidative damage, thereby as a potential natural drug to treat neurodegenerative disorders.


Assuntos
Antioxidantes , Astragalus propinquus , Drosophila melanogaster , Doenças Neurodegenerativas , Estresse Oxidativo , Animais , Estresse Oxidativo/efeitos dos fármacos , Astragalus propinquus/química , Drosophila melanogaster/efeitos dos fármacos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Antioxidantes/farmacologia , Fármacos Neuroprotetores/farmacologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Extratos Vegetais/farmacologia , Animais Geneticamente Modificados , Medicamentos de Ervas Chinesas/farmacologia , Peróxido de Hidrogênio , Peptídeos beta-Amiloides/metabolismo
5.
Life Sci Alliance ; 7(9)2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38876797

RESUMO

Calcium is critical for regulating the waveform of motile cilia and flagella. Calaxin is currently the only known molecule involved in the calcium-dependent regulation in ascidians. We have recently shown that Calaxin stabilizes outer arm dynein (OAD), and the knockout of Calaxin results in primary ciliary dyskinesia phenotypes in vertebrates. However, from the knockout experiments, it was not clear which functions depend on calcium and how Calaxin regulates the waveform. To address this question, here, we generated transgenic zebrafish expressing a mutant E130A-Calaxin deficient in calcium binding. E130A-Calaxin restored the OAD reduction of calaxin -/- sperm and the abnormal movement of calaxin -/- left-right organizer cilia, showing that Calaxin's stabilization of OADs is calcium-independent. In contrast, our quantitative analysis of E130A-Calaxin sperms showed that the calcium-induced asymmetric beating was not restored, linking Calaxin's calcium-binding ability with an asymmetric flagellar beating for the first time. Our data show that Calaxin is a calcium-dependent regulator of the ciliary beating and a calcium-independent OAD stabilizer.


Assuntos
Animais Geneticamente Modificados , Cálcio , Dineínas , Espermatozoides , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Masculino , Cálcio/metabolismo , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Dineínas/metabolismo , Dineínas/genética , Cílios/metabolismo , Flagelos/metabolismo , Flagelos/fisiologia , Motilidade dos Espermatozoides/genética , Motilidade dos Espermatozoides/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética
6.
Xenotransplantation ; 31(3): e12872, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38924560

RESUMO

Attack of donor tissues by pre-formed anti-pig antibodies is well known to cause graft failure in xenotransplantation. Genetic engineering of porcine donors to eliminate targets of these pre-formed antibodies coupled with advances in immunosuppressive medicines have now made it possible to achieve extended survival in the pre-clinical pig-to-non-human primate model. Despite these improvements, antibodies remain a risk over the lifetime of the transplant, and many patients continue to have pre-formed donor-specific antibodies even to highly engineered pigs. While therapeutics exist that can help mitigate the detrimental effects of antibodies, they act broadly potentially dampening beneficial immunity. Identifying additional xenoantigens may enable more targeted approaches, such as gene editing, to overcome these challenges by further eliminating antibody targets on donor tissue. Because we have found that classical class I swine leukocyte antigens are targets of human antibodies, we now examine whether related pig proteins may also be targeted by human antibodies. We show here that non-classical class I swine leukocyte proteins (SLA-6, -7, -8) can be expressed at the surface of mammalian cells and act as antibody targets.


Assuntos
Antígenos Heterófilos , Antígenos de Histocompatibilidade Classe I , Transplante Heterólogo , Animais , Suínos , Transplante Heterólogo/métodos , Antígenos Heterófilos/imunologia , Humanos , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Rejeição de Enxerto/imunologia , Animais Geneticamente Modificados
7.
Sci Rep ; 14(1): 12826, 2024 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-38834813

RESUMO

Lamin A/C gene (LMNA) mutations contribute to severe striated muscle laminopathies, affecting cardiac and skeletal muscles, with limited treatment options. In this study, we delve into the investigations of five distinct LMNA mutations, including three novel variants and two pathogenic variants identified in patients with muscular laminopathy. Our approach employs zebrafish models to comprehensively study these variants. Transgenic zebrafish expressing wild-type LMNA and each mutation undergo extensive morphological profiling, swimming behavior assessments, muscle endurance evaluations, heartbeat measurement, and histopathological analysis of skeletal muscles. Additionally, these models serve as platform for focused drug screening. We explore the transcriptomic landscape through qPCR and RNAseq to unveil altered gene expression profiles in muscle tissues. Larvae of LMNA(L35P), LMNA(E358K), and LMNA(R453W) transgenic fish exhibit reduced swim speed compared to LMNA(WT) measured by DanioVision. All LMNA transgenic adult fish exhibit reduced swim speed compared to LMNA(WT) in T-maze. Moreover, all LMNA transgenic adult fish, except LMNA(E358K), display weaker muscle endurance than LMNA(WT) measured by swimming tunnel. Histochemical staining reveals decreased fiber size in all LMNA mutations transgenic fish, excluding LMNA(WT) fish. Interestingly, LMNA(A539V) and LMNA(E358K) exhibited elevated heartbeats. We recognize potential limitations with transgene overexpression and conducted association calculations to explore its effects on zebrafish phenotypes. Our results suggest lamin A/C overexpression may not directly impact mutant phenotypes, such as impaired swim speed, increased heart rates, or decreased muscle fiber diameter. Utilizing LMNA zebrafish models for drug screening, we identify L-carnitine treatment rescuing muscle endurance in LMNA(L35P) and creatine treatment reversing muscle endurance in LMNA(R453W) zebrafish models. Creatine activates AMPK and mTOR pathways, improving muscle endurance and swim speed in LMNA(R453W) fish. Transcriptomic profiling reveals upstream regulators and affected genes contributing to motor dysfunction, cardiac anomalies, and ion flux dysregulation in LMNA mutant transgenic fish. These findings faithfully mimic clinical manifestations of muscular laminopathies, including dysmorphism, early mortality, decreased fiber size, and muscle dysfunction in zebrafish. Furthermore, our drug screening results suggest L-carnitine and creatine treatments as potential rescuers of muscle endurance in LMNA(L35P) and LMNA(R453W) zebrafish models. Our study offers valuable insights into the future development of potential treatments for LMNA-related muscular laminopathy.


Assuntos
Animais Geneticamente Modificados , Carnitina , Creatina , Lamina Tipo A , Músculo Esquelético , Mutação , Peixe-Zebra , Animais , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/efeitos dos fármacos , Creatina/metabolismo , Carnitina/metabolismo , Modelos Animais de Doenças , Laminopatias/genética , Laminopatias/metabolismo , Natação , Transcriptoma , Humanos
8.
Development ; 151(13)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38828852

RESUMO

The cellular and genetic networks that contribute to the development of the zeugopod (radius and ulna of the forearm, tibia and fibula of the leg) are not well understood, although these bones are susceptible to loss in congenital human syndromes and to the action of teratogens such as thalidomide. Using a new fate-mapping approach with the Chameleon transgenic chicken line, we show that there is a small contribution of SHH-expressing cells to the posterior ulna, posterior carpals and digit 3. We establish that although the majority of the ulna develops in response to paracrine SHH signalling in both the chicken and mouse, there are differences in the contribution of SHH-expressing cells between mouse and chicken as well as between the chicken ulna and fibula. This is evidence that, although zeugopod bones are clearly homologous according to the fossil record, the gene regulatory networks that contribute to their development and evolution are not fixed.


Assuntos
Animais Geneticamente Modificados , Galinhas , Proteínas Hedgehog , Animais , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Galinhas/genética , Camundongos , Evolução Biológica , Embrião de Galinha , Ulna , Regulação da Expressão Gênica no Desenvolvimento , Fíbula/metabolismo , Rádio (Anatomia)/metabolismo , Humanos , Extremidades/embriologia
9.
Learn Mem ; 31(5)2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38862177

RESUMO

Associative learning enables the adaptive adjustment of behavioral decisions based on acquired, predicted outcomes. The valence of what is learned is influenced not only by the learned stimuli and their temporal relations, but also by prior experiences and internal states. In this study, we used the fruit fly Drosophila melanogaster to demonstrate that neuronal circuits involved in associative olfactory learning undergo restructuring during extended periods of low-caloric food intake. Specifically, we observed a decrease in the connections between specific dopaminergic neurons (DANs) and Kenyon cells at distinct compartments of the mushroom body. This structural synaptic plasticity was contingent upon the presence of allatostatin A receptors in specific DANs and could be mimicked optogenetically by expressing a light-activated adenylate cyclase in exactly these DANs. Importantly, we found that this rearrangement in synaptic connections influenced aversive, punishment-induced olfactory learning but did not impact appetitive, reward-based learning. Whether induced by prolonged low-caloric conditions or optogenetic manipulation of cAMP levels, this synaptic rearrangement resulted in a reduction of aversive associative learning. Consequently, the balance between positive and negative reinforcing signals shifted, diminishing the ability to learn to avoid odor cues signaling negative outcomes. These results exemplify how a neuronal circuit required for learning and memory undergoes structural plasticity dependent on prior experiences of the nutritional value of food.


Assuntos
Drosophila melanogaster , Corpos Pedunculados , Plasticidade Neuronal , Animais , Corpos Pedunculados/fisiologia , Corpos Pedunculados/metabolismo , Drosophila melanogaster/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios Dopaminérgicos/fisiologia , Neurônios Dopaminérgicos/metabolismo , Ingestão de Alimentos/fisiologia , Optogenética , Aprendizagem por Associação/fisiologia , Olfato/fisiologia , Percepção Olfatória/fisiologia , Recompensa , Animais Geneticamente Modificados
10.
Neural Dev ; 19(1): 11, 2024 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-38909268

RESUMO

The complex morphology of neurons requires precise control of their microtubule cytoskeleton. This is achieved by microtubule-associated proteins (MAPs) that regulate the assembly and stability of microtubules, and transport of molecules and vesicles along them. While many of these MAPs function in all cells, some are specifically or predominantly involved in regulating microtubules in neurons. Here we use the sea anemone Nematostella vectensis as a model organism to provide new insights into the early evolution of neural microtubule regulation. As a cnidarian, Nematostella belongs to an outgroup to all bilaterians and thus occupies an informative phylogenetic position for reconstructing the evolution of nervous system development. We identified an ortholog of the microtubule-binding protein doublecortin-like kinase (NvDclk1) as a gene that is predominantly expressed in neurons and cnidocytes (stinging cells), two classes of cells belonging to the neural lineage in cnidarians. A transgenic NvDclk1 reporter line revealed an elaborate network of neurite-like processes emerging from cnidocytes in the tentacles and the body column. A transgene expressing NvDclk1 under the control of the NvDclk1 promoter suggests that NvDclk1 localizes to microtubules and therefore likely functions as a microtubule-binding protein. Further, we generated a mutant for NvDclk1 using CRISPR/Cas9 and show that the mutants fail to generate mature cnidocytes. Our results support the hypothesis that the elaboration of programs for microtubule regulation occurred early in the evolution of nervous systems.


Assuntos
Quinases Semelhantes a Duplacortina , Neurônios , Anêmonas-do-Mar , Animais , Anêmonas-do-Mar/embriologia , Anêmonas-do-Mar/citologia , Anêmonas-do-Mar/genética , Neurônios/metabolismo , Neurônios/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Microtúbulos/metabolismo , Neurogênese/fisiologia , Animais Geneticamente Modificados , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética
11.
J Vis Exp ; (208)2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38912770

RESUMO

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope. Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.


Assuntos
Drosophila , Técnicas de Transferência de Genes , Microinjeções , Animais , Microinjeções/métodos , Técnicas de Transferência de Genes/instrumentação , Drosophila/genética , Drosophila/embriologia , Plasmídeos/genética , Plasmídeos/administração & dosagem , Embrião não Mamífero , Animais Geneticamente Modificados , Integrases/genética
12.
J Cell Biol ; 223(9)2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-38913324

RESUMO

Here, we report the generation of a transgenic Lifeact-EGFP quail line for the investigation of actin organization and dynamics during morphogenesis in vivo. This transgenic avian line allows for the high-resolution visualization of actin structures within the living embryo, from the subcellular filaments that guide cell shape to the supracellular assemblies that coordinate movements across tissues. The unique suitability of avian embryos to live imaging facilitates the investigation of previously intractable processes during embryogenesis. Using high-resolution live imaging approaches, we present the dynamic behaviors and morphologies of cellular protrusions in different tissue contexts. Furthermore, through the integration of live imaging with computational segmentation, we visualize cells undergoing apical constriction and large-scale actin structures such as multicellular rosettes within the neuroepithelium. These findings not only enhance our understanding of tissue morphogenesis but also demonstrate the utility of the Lifeact-EGFP transgenic quail as a new model system for live in vivo investigations of the actin cytoskeleton.


Assuntos
Citoesqueleto de Actina , Actinas , Animais Geneticamente Modificados , Proteínas de Fluorescência Verde , Codorniz , Animais , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Actinas/metabolismo , Actinas/genética , Citoesqueleto de Actina/metabolismo , Morfogênese , Embrião não Mamífero/metabolismo
13.
Sci Rep ; 14(1): 14332, 2024 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-38906973

RESUMO

Spinocerebellar ataxia type 7 (SCA7) is a progressive neurodegenerative disorder resulting from abnormal expansion of an uninterrupted polyglutamine (polyQ) repeat in its disease protein, ataxin-7 (ATXN7). ATXN7 is part of Spt-Ada-Gcn5 acetyltransferase (SAGA), an evolutionarily conserved transcriptional coactivation complex with critical roles in chromatin remodeling, cell signaling, neurodifferentiation, mitochondrial health and autophagy. SCA7 is dominantly inherited and characterized by genetic anticipation and high repeat-length instability. Patients with SCA7 experience progressive ataxia, atrophy, spasticity, and blindness. There is currently no cure for SCA7, and therapies are aimed at alleviating symptoms to increase quality of life. Here, we report novel Drosophila lines of SCA7 with polyQ repeats in wild-type and human disease patient range. We find that ATXN7 expression has age- and polyQ repeat length-dependent reduction in fruit fly survival and retinal instability, concomitant with increased ATXN7 protein aggregation. These new lines will provide important insight on disease progression that can be used in the future to identify therapeutic targets for SCA7 patients.


Assuntos
Ataxina-7 , Modelos Animais de Doenças , Peptídeos , Ataxias Espinocerebelares , Animais , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia , Ataxias Espinocerebelares/metabolismo , Ataxina-7/genética , Ataxina-7/metabolismo , Humanos , Peptídeos/metabolismo , Peptídeos/genética , Drosophila/genética , Animais Geneticamente Modificados , Progressão da Doença , Drosophila melanogaster/genética , Retina/metabolismo , Retina/patologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo
14.
Dev Comp Immunol ; 158: 105208, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38834141

RESUMO

Interferon regulatory factors (IRFs) are transcription factors involved in immune responses, such as pathogen response regulation, immune cell growth, and differentiation. IRFs are necessary for the synthesis of type I interferons through a signaling cascade when pathogen recognition receptors identify viral DNA or RNA. We discovered that irf3 is expressed in the early embryonic stages and in all immune organs of adult zebrafish. We demonstrated the antiviral immune mechanism of Irf3 against viral hemorrhagic septicemia virus (VHSV) using CRISPR/Cas9-mediated knockout zebrafish (irf3-KO). In this study, we used a truncated Irf3 protein, encoded by irf3 with a 10 bp deletion, for further investigation. Upon VHSV injection, irf3-KO zebrafish showed dose-dependent high and early mortality compared with zebrafish with the wild-type Irf3 protein (WT), confirming the antiviral activity of Irf3. Based on the results of expression analysis of downstream genes upon VHSV challenge, we inferred that Irf3 deficiency substantially affects the expression of ifnphi1 and ifnphi2. However, after 5 days post infection (dpi), ifnphi3 expression was not significantly altered in irf3-KO compared to that in WT, and irf7 transcription showed a considerable increase in irf3-KO after 5 dpi, indicating irf7's control over ifnphi3 expression. The significantly reduced expression of isg15, viperin, mxa, and mxb at 3 dpi also supported the effect of Irf3 deficiency on the antiviral activity in the early stage of infection. The higher mortality in irf3-KO zebrafish than in WT might be due to an increased inflammation and tissue damage that occurs in irf3-KO because of delayed immune response. Our results suggest that Irf3 plays a role in antiviral immunity of zebrafish by modulating critical immune signaling molecules and regulating antiviral immune genes.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Septicemia Hemorrágica Viral , Fator Regulador 3 de Interferon , Novirhabdovirus , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Novirhabdovirus/fisiologia , Novirhabdovirus/imunologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Septicemia Hemorrágica Viral/imunologia , Septicemia Hemorrágica Viral/genética , Septicemia Hemorrágica Viral/virologia , Animais Geneticamente Modificados , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Doenças dos Peixes/genética , Imunidade Inata/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/virologia , Modelos Animais de Doenças , Interferons
15.
Sci Adv ; 10(23): eadn6603, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38838146

RESUMO

Standard zebrafish transgenesis involves random transgene integration with resource-intensive screening. While phiC31 integrase-based attP/attB recombination has streamlined transgenesis in mice and Drosophila, validated attP-based landing sites for universal applications are lacking in zebrafish. Here, we developed phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET) as transgenesis approach, with two attP landing sites pIGLET14a and pIGLET24b from well-validated Tol2 transgenes. Both sites facilitate diverse transgenesis applications including reporters and Cre/loxP transgenes. The pIGLET14a and pIGLET24b landing sites consistently yield 25 to 50% germline transmission, substantially reducing the resources needed for transgenic line generation. Transgenesis into these sites enables reproducible expression patterns in F0 zebrafish embryos for enhancer discovery and testing of gene regulatory variants. Together, our new landing sites streamline targeted, reproducible zebrafish transgenesis as a robust platform for various applications while minimizing the workload for generating transgenic lines.


Assuntos
Animais Geneticamente Modificados , Técnicas de Transferência de Genes , Transgenes , Peixe-Zebra , Animais , Peixe-Zebra/genética , Integrases/genética , Integrases/metabolismo , Sítios de Ligação Microbiológicos/genética
16.
Alzheimers Res Ther ; 16(1): 123, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38849926

RESUMO

BACKGROUND: Recent reports suggest that amyloid beta (Aß) peptides can exhibit prion-like pathogenic properties. Transmission of Aß peptide and the development of associated pathologies after surgeries with contaminated instruments and intravenous or intracerebral inoculations have now been reported across fish, rodents, primates, and humans. This raises a worrying prospect of Aß peptides also having other characteristics typical of prions, such as evasion of the digestive process. We asked if such transmission of Aß aggregates via ingestion was possible. METHODS: We made use of a transgenic Drosophila melanogaster line expressing human Aß peptide prone to aggregation. Fly larvae were fed to adult zebrafish under two feeding schemes. The first was a short-term, high-intensity scheme over 48 h to determine transmission and retention in the gut. The second, long-term scheme specifically examined retention and accumulation in the brain. The gut and brain tissues were examined by histology, western blotting, and mass spectrometric analyses. RESULTS: None of the analyses could detect Aß aggregates in the guts of zebrafish following ingestion, despite being easily detectable in the feed. Additionally, there was no detectable accumulation of Aß in the brain tissue or development of associated pathologies after prolonged feeding. CONCLUSIONS: While human Aß aggregates do not appear to be readily transmissible by ingestion across species, two prospects remain open. First, this mode of transmission, if occurring, may stay below a detectable threshold and may take much longer to manifest. A second possibility is that the human Aß peptide is not able to trigger self-propagation or aggregation in other species. Either possibility requires further investigation, taking into account the possibility of such transmission from agricultural species used in the food industry.


Assuntos
Peptídeos beta-Amiloides , Animais Geneticamente Modificados , Encéfalo , Drosophila melanogaster , Peixe-Zebra , Animais , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Humanos , Ingestão de Alimentos/fisiologia , Larva , Agregados Proteicos
17.
Elife ; 132024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38874379

RESUMO

Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this TGFb Induced Enhancer driving green fluorescent protein (TIE:EGFP). TIE:EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of advanced melanomas. Single-cell RNA-sequencing revealed that TIE:EGFP+ melanoma cells down-regulated interferon response while up-regulating a novel set of chronic TGFb target genes. ChIP-sequencing demonstrated that AP-1 factor binding is required for activation of chronic TGFb response. Overexpression of SATB2, a chromatin remodeler associated with tumor spreading, showed activation of TGFb signaling in early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages localize to TIE:EGFP+ regions and preferentially phagocytose TIE:EGFP+ melanoma cells compared to TIE:EGFP- melanoma cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.


Assuntos
Animais Geneticamente Modificados , Melanoma , Transdução de Sinais , Peixe-Zebra , Melanoma/genética , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/patologia , Animais , Humanos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Fator de Crescimento Transformador beta1/metabolismo , Linhagem Celular Tumoral , Genes Reporter , Fator de Crescimento Transformador beta/metabolismo , Regulação Neoplásica da Expressão Gênica
18.
Nat Commun ; 15(1): 4983, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862555

RESUMO

Engineered sex ratio distorters (SRDs) have been proposed as a powerful component of genetic control strategies designed to suppress harmful insect pests. Two types of CRISPR-based SRD mechanisms have been proposed: X-shredding, which eliminates X-bearing sperm, and X-poisoning, which eliminates females inheriting disrupted X-chromosomes. These differences can have a profound impact on the population dynamics of SRDs when linked to the Y-chromosome: an X-shredder is invasive, constituting a classical meiotic Y-drive, whereas X-poisoning is self-limiting, unable to invade but also insulated from selection. Here, we establish X-poisoning strains in the malaria vector Anopheles gambiae targeting three X-linked genes during spermatogenesis, resulting in male bias. We find that sex distortion is primarily driven by a loss of X-bearing sperm, with limited evidence for postzygotic lethality of female progeny. By leveraging a Drosophila melanogaster model, we show unambiguously that engineered SRD traits can operate differently in these two insects. Unlike X-shredding, X-poisoning could theoretically operate at early stages of spermatogenesis. We therefore explore premeiotic Cas9 expression to target the mosquito X-chromosome. We find that, by pre-empting the onset of meiotic sex chromosome inactivation, this approach may enable the development of Y-linked SRDs if mutagenesis of spermatogenesis-essential genes is functionally balanced.


Assuntos
Anopheles , Drosophila melanogaster , Tecnologia de Impulso Genético , Razão de Masculinidade , Espermatogênese , Cromossomo X , Animais , Masculino , Feminino , Anopheles/genética , Cromossomo X/genética , Drosophila melanogaster/genética , Tecnologia de Impulso Genético/métodos , Espermatogênese/genética , Mosquitos Vetores/genética , Genes Ligados ao Cromossomo X , Sistemas CRISPR-Cas , Espermatozoides/metabolismo , Animais Geneticamente Modificados
19.
Xenotransplantation ; 31(3): e12865, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38853364

RESUMO

Recent clinical xenotransplantation and human decedent studies demonstrate that clinical hyperacute rejection of genetically engineered porcine organs can be reliably avoided but that antibody mediated rejection (AMR) continues to limit graft survival. We previously identified porcine glycans and proteins which are immunogenic after cardiac xenotransplantation in non-human primates, but the clinical immune response to antigens present in glycan depleted triple knockout (TKO) donor pigs is poorly understood. In this study we use fluorescence barcoded human embryonic kidney cells (HEK) and HEK cell lines expressing porcine glycans (Gal and SDa) or proteins (tetraspanin-29 [CD9], membrane cofactor protein [CD46], protectin, membrane attack complex inhibition factor [CD59], endothelial cell protein C receptor, and Annexin A2) to screen antibody reactivity in human serum from 160 swine veterinarians, a serum source with potential occupational immune challenge from porcine tissues and pathogens. High levels of anti-Gal IgM were present in all samples and lower levels of anti-SDa IgM were present in 41% of samples. IgM binding to porcine proteins, primarily CD9 and CD46, previously identified as immunogenic in pig to non-human primate cardiac xenograft recipients, was detected in 28 of the 160 swine veterinarian samples. These results suggest that barcoded HEK cell lines expressing porcine protein antigens can be useful for screening human patient serum. A comprehensive analysis of sera from clinical xenotransplant recipients to define a panel of commonly immunogenic porcine antigens will likely be necessary to establish an array of porcine non-Gal antigens for effective monitoring of patient immune responses and allow earlier therapies to reverse AMR.


Assuntos
Rejeição de Enxerto , Transplante Heterólogo , Animais , Transplante Heterólogo/métodos , Humanos , Suínos , Rejeição de Enxerto/imunologia , Células HEK293 , Médicos Veterinários , Polissacarídeos/imunologia , Animais Geneticamente Modificados , Anticorpos Heterófilos/imunologia , Anticorpos Heterófilos/sangue , Xenoenxertos/imunologia , Imunoglobulina M/imunologia , Imunoglobulina M/sangue
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