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1.
Food Res Int ; 160: 111696, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36076399

RESUMO

Alzheimer's disease (AD) is one of the most severe neurodegenerative disorders. Recently, there is no effective treatment drug for AD. Morus nigra (M. nigra) is a black mulberry and widely distributed fruit in the Moraceae family with various undiscovered biological activities. The study aimed to investigate the potential anti-AD effect of M. nigra. Mulberry fruit extract (MF) was obtained from M. nigra and treated up to 1.00 mg/mL on transgenic AD Caenorhabditis elegans (C. elegans) models. MF inhibited Amyloid-ß (Aß)-induced paralysis symptoms by about 55.65 %, reduced Aß accumulation more than 50 % via immunoblotting, and suppressed over-sensitivity to exogenous serotonin in C. elegans. Furthermore, MF decreased the Aß oligomeric depositions in worm CL2006. MF activated the DAF-16 nuclear translocation and its downstream SOD-3 and GST-4. AD is a major age-related disorder. Therefore, MF treated for an aging test and proved to be expanded the lifespan of the worms up to 34.7 %. Besides, we have evaluated the MF in vivo antioxidative properties, where MF reduced reactive oxygen species (ROS) generations in C. elegans and remitted the activation of HSP-16.2 induced by the oxidative action of Juglone. Gene knockout and extended the lifespan of AD worms. However, RNA interference (RNAi) successfully silenced the daf-16 on the Aß phenotypic paralysis proved by MF effect. Our results indicate that MF alleviates AD-Like symptoms by activating the DAF-16 insulin signal pathway in C. elegans. Therefore, this MF study may provide new insights for mulberry application in safe AD treatment and clinical study.


Assuntos
Doença de Alzheimer , Proteínas de Caenorhabditis elegans , Morus , Peptídeos beta-Amiloides , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/farmacologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/farmacologia , Frutas/metabolismo , Insulina/metabolismo , Morus/metabolismo , Estresse Oxidativo , Paralisia , Extratos Vegetais/metabolismo , Transdução de Sinais
2.
Zhonghua Yi Xue Za Zhi ; 102(33): 2619-2623, 2022 Sep 06.
Artigo em Chinês | MEDLINE | ID: mdl-36058688

RESUMO

Objective: To construct zebrafish models for the screening of intracranial hemorrhage (ICH) associated genes. Methods: ICH zebrafish models were constructed through morpholino oligonucleotides (MOs) technique and microinjection technique, and multiple verification was performed from macro and micro perspectives. First, the normal wild-type AB strain zebrafish injected with control MO was used as the control group, and AB zebrafish embryos microinjected with MOs of genes related to development of neural crest-derived cells (NCDCs) were used as the study group, such as col8a1 MO, tfap2α MO, msx1a MO, msx2 MO, and dkk1a MO. Preliminary verification of the model was conducted under a white-light optical microscope. Then, the model was verified by Tg (flk1: gfp; gata1: dsRed) double transgenic zebrafish, with vascular endothelial cells labeled by green fluorescent protein (GFP) and red blood cell labeled by fluorescent protein (dsRed), and thus the location of cerebral hemorrhage can be observed more clearly. Specifically, zebrafish embryos were microinjected with Control MO as the control group and those microinjected with col8a1 MO as the study group. Then the embryos were cultured until 48 hours post-fertilization to observe the leakage of red blood cells under the confocal laser scanning microscope. Finally, Tg (flk1: gfp) transgenic zebrafish was used to verify the model based on the blood-brain barrier (BBB). Through the leakage of dextran-rhodamine and DAPI dyes, the destruction of BBB and the occurrence of cerebral hemorrhage in zebrafish were further clarified, and quantitative statistics were carried out to verify the relationship between NCDCs development related genes and cerebral hemorrhage phenotype, which proved that the modeling was effective. Results: The zebrafish with col8a1, tfap2α, and msx1 mutations in the study group had apparent ICH compared with wildtype zebrafish, and the prevalence of ICH was 18.18% (52/286), 23.04% (62/251), and 35.94% (23/64), respectively. While, the zebrafish with msx2 and dkk1a mutations rarely had ICH, with the ICH prevalence of 1.03% (1/97) and 1.15% (1/87), respectively. The prevalence of red blood cells leakage in Tg (flk1:gfp; gata1:dsred) double transgenic zebrafish injected with Control Mo and col8a1 Mo was 0.37% (1/273) and 18.18% (52/286) (P<0.001). The number of DAPI positive nuclei of Tg (flk1: gfp) transgenic zebrafish injected with Control Mo and col8a1 Mo was 10.05±5.27 and 60.35±3.96 (P<0.001), and the fluorescent intensity of midbrain parenchymal induced by dextran-rhodamin leakage was 2.54±4.70 and 5.13±3.52 (P<0.001). Conclusion: This study successfully constructs the ICH zebrafish models, and ICH-related genes are screened out, such as col8a1, tfap2α, msx1, and so on.


Assuntos
Células Endoteliais , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Hemorragia Cerebral , Dextranos , Proteínas de Fluorescência Verde , Peixe-Zebra/genética
3.
Int J Mol Sci ; 23(17)2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36077351

RESUMO

Growth factors, including fibroblast growth factor-7 (FGF-7), are a group of proteins that stimulate various cellular processes and are often used with carriers to prevent the rapid loss of their activities. Sericin with great biocompatibility has been investigated as a proteinaceous carrier to enhance the stability of incorporated proteins. The difficulties in obtaining intact sericin from silkworm cocoons and the handling of growth factors with poor stability necessitate an efficient technique to incorporate the protein into a sericin-based biomaterial. Here, we report the generation of a transgenic silkworm line simultaneously expressing and incorporating FGF-7 into cocoon shells containing almost exclusively sericin. Growth-factor-functionalized sericin cocoon shells requiring simple lyophilization and pulverization processes were successfully used to induce the proliferation and migration of keratinocytes. Moreover, FGF-7 incorporated into sericin-cocoon powder exhibited remarkable stability, with more than 70% of bioactivity being retained after being stored as a suspension at 25 °C for 3 months. Transgenic sericin-cocoon powder was used to continuously supply biologically active FGF-7 to generate a three-dimensionally cultured keratinocyte model in vitro. The outcomes of this study propound a feasible approach to producing cytokine-functionalized sericin materials that are ready to use for cell cultivation.


Assuntos
Bombyx , Sericinas , Animais , Animais Geneticamente Modificados , Materiais Biocompatíveis/farmacologia , Bioengenharia , Bombyx/genética , Bombyx/metabolismo , Queratinócitos/metabolismo , Pós , Sericinas/metabolismo , Sericinas/farmacologia
4.
Int J Mol Sci ; 23(17)2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36077432

RESUMO

Alzheimer's disease (AD) is one of the leading causes of dementia. As the first common neurodegenerative disease, there are no effective drugs that can reverse the progression. The present study is to report the anti-AD effect of cryptotanshinone (CTS), a natural product isolated from Salvia castanea. It is found that it can alleviate AD-like features associated with Aß1-42 toxicity in muscle cells as well as neuronal cells of Caenorhabditis elegans (C. elegans). Further studies showed that CTS reduced the level of reactive oxygen species (ROS) in nematodes, up-regulated the expression of sod-3, and enhanced superoxide dismutase activity. Cryptotanshinone reduced the level of Aß monomers and highly toxic oligomers in C. elegans while inhibiting the abnormal aggregation of polyglutamine protein. In addition, CTS upregulated the expression of hsp-16.2 and downregulated the expression of ace-2. These results suggested that CTS could alleviate oxidative stress and reduce the level of abnormally aggregated proteins and has the potential to be developed as an anti-AD drug candidate.


Assuntos
Doença de Alzheimer , Proteínas de Caenorhabditis elegans , Doenças Neurodegenerativas , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Estresse Oxidativo , Fenantrenos , Espécies Reativas de Oxigênio/metabolismo
5.
Front Immunol ; 13: 974014, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36091009

RESUMO

White spot syndrome is an epidemic disease caused by the highly contagious and lethal white spot syndrome virus (WSSV), resulting in huge economic losses to the global aquaculture industry. VP28 is the main structural protein in the capsule of WSSV and is important in the early stage of infection. Under an excitation wavelength of 548 nm, the mOrange fluorescent protein releases a 562 nm emission wavelength, which is different from the autofluorescence of cyanobacteria. Therefore, using this characteristic combined with the receptor system of Synechococcus elongatus PCC 7942, we constructed transgenic S. elongatus to express the recombinant protein VP28-mOrange. In addition, PCR and western blotting were used to confirm the stable expression of the target gene in cyanobacteria. Using mOrange tracer features, we explored the recombinant protein VP28-mOrange in the metabolic cycle of young Litopenaeus Vannamei after feeding. After the young shrimp had stopped consuming transgenic cyanobacteria, the 24 to 33 h fluorescence signal in the intestine was very weak, and almost disappeared after 36 h. We explored the protective effect of transgenic vp28-mOrange S. elongatus within 48 h of being ingested by L. vannamei and set WSSV challenges at 2, 12, 24, and 48 h post-immunization. However, the survival rate of L. vannamei decreased as the time of the WSSV challenge increased. The survival rate on the seventh day was 81%, 52%, 45.5%, and 33.3% for shrimps challenged for 2, 12, 24, and 48 h, respectively. Enzyme activity can also support this conjecture, the enzyme activity indexes of the experimental groups were significantly reduced compared to positive and wild-type controls. Therefore, this immune agent functioned as a preventive agent. Compared with the traditional method, this method was easy to detect and can visualize the digestion of transgenic cyanobacteria in the Litopenaeus vannamei intestine.


Assuntos
Penaeidae , Vírus da Síndrome da Mancha Branca 1 , Animais , Animais Geneticamente Modificados , Proteínas Luminescentes , Proteínas Recombinantes/genética , Synechococcus , Proteínas do Envelope Viral , Vírus da Síndrome da Mancha Branca 1/genética
6.
Cells ; 11(15)2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35954224

RESUMO

Farm animal salivary glands hold great potential as efficient bioreactors for production of human therapeutic proteins. Nerve growth factor (NGF) is naturally expressed in animal salivary glands and has been approved for human clinical treatment. This study aims to employ transgenic (TG) pig salivary gland as bioreactors for efficient synthesis of human NGF (hNGF). hNGF-TG pigs were generated by cloning in combination with piggyBac transposon-mediated gene transfer. These hNGF-TG pigs specifically expressed hNGF protein in their salivary glands and secreted it at high levels into saliva. Surgical and nonsurgical approaches were developed to efficiently collect saliva from hNGF-TG pigs. hNGF protein was successfully purified from collected saliva and was verified to be biologically active. In an additional step, the double-transgenic pigs, where the endogenous porcine NGF (pNGF) gene was replaced by another copy of hNGF transgene, were created by cloning combined with CRISPR/Cas9-mediated homologous recombination. These double-transgenic pigs expressed hNGF but not pNGF, thus avoiding possible "contamination" of hNGF with pNGF protein during purification. In conclusion, TG pig salivary glands can be used as robust bioreactors for a large-scale synthesis of functional hNGF or other valuable proteins. This new animal pharming method will benefit both human health and biomedicine.


Assuntos
Fator de Crescimento Neural , Glândulas Salivares , Animais , Animais Geneticamente Modificados , Reatores Biológicos , Humanos , Fator de Crescimento Neural/metabolismo , Glândulas Salivares/metabolismo , Suínos , Transgenes
7.
J Agric Food Chem ; 70(32): 10011-10021, 2022 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-35917150

RESUMO

Methylglyoxal (MG) is a precursor of advanced glycation end products usually generated during cooking. The high level of MG in the brain is correlated to the pathogenesis of Alzheimer's disease (AD). However, it is not clear if MG consumed through the diet can cause AD-related toxicity. Herein, the Caenorhabditis elegans (C. elegans) AD model was used to investigate the neurotoxicity after long-term MG exposure at dietary levels. The results showed that C. elegans locomotive behaviors were significantly decreased after 0.1, 0.5, and 1 mM MG exposure (p < 0.001). In amyloid ß (Aß)-expressing transgenic C. elegans strains, 0.5 mM MG significantly promoted Aß accumulation by around 50% in day-8 CL2006 (p < 0.001), enhanced paralysis in CL4176 (p < 0.001) and CL2006 (p < 0.01), and made CL2355 around 17% more vulnerable to 5-HT, indicating impaired serotonin reuptake (p < 0.05). Additionally, 0.5 mM MG significantly increased the reactive oxygen species level (p < 0.001) by inhibiting the expression of stress-response genes including sod-3, gst-4, and hsp-16.2 in day-8 aged worms. Moreover, the autophagic pathway was disrupted through lgg-1, vps-34, and bec-1 expression after MG exposure and Aß accumulation. Treatment with the citrus flavonoid nobiletin reduced the MG-induced toxicity (p < 0.001). Overall, these findings imply that it is possible to exacerbate AD pathogenesis by MG exposure through the diet.


Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Geneticamente Modificados , Autofagia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Estresse Oxidativo , Fragmentos de Peptídeos/metabolismo , Aldeído Pirúvico/metabolismo , Aldeído Pirúvico/toxicidade
8.
Open Biol ; 12(8): 220104, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35946311

RESUMO

Cilia are organelles for cellular signalling and motility. Mutations affecting ciliary function are also associated with cilia-related disorders (ciliopathies). The identification of cilia markers is critical for studying their function at the cellular level. Due to the lack of a conserved, short ciliary localization motif, the full-length ARL13b or 5HT6 proteins are normally used for cilia labelling. Overexpression of these genes, however, can affect the function of cilia, leading to artefacts in cilia studies. Here, we show that Nephrocystin-3 (Nphp3) is highly conserved among vertebrates and demonstrate that the N-terminal truncated peptide of zebrafish Nphp3 can be used as a gratuitous cilia-specific marker. To visualize the dynamics of cilia in vivo, we generated a stable transgenic zebrafish Tg (ß-actin: nphp3N-mCherry)sx1001. The cilia in multiple cell types are efficiently labelled by the encoded fusion protein from embryonic stages to adulthood, without any developmental and physiological defects. We show that the line allows live imaging of ciliary dynamics and trafficking of cilia proteins, such as Kif7 and Smo, key regulators of the Hedgehog signalling pathway. Thus, we have generated an effective new tool for in vivo cilia studies that will help shed further light on the roles of these important organelles.


Assuntos
Cílios , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Cílios/genética , Cílios/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Mutação
9.
Gene Expr Patterns ; 45: 119269, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35970322

RESUMO

BACKGROUND: Expression level of EMX2 plays an important role in the development of nervous system and cancers. CNE2.04, a conserved enhancer downstream of emx2, drives fluorescent protein expression in the similar pattern of emx2. METHODS: CNE2.04 truncated or motif-mutated transgenic reporter plasmids were constructed and injected into the zebrafish fertilized egg with Tol2 mRNA at the unicellular stage of zebrafish eggs. The green fluorescence expression patterns were observed at 24, 48, and 72 hpf, and the fluorescence rates of different tissues were counted at 48 hpf. RESULTS: Compared to CNE2.04, CNE2.04-R400 had comparable enhancer activity, while the tissue specificity of CNE2.04-L400 was obviously changed. Motif CCCCTC mutation obviously changed the enhancer activity, while motif CCGCTC mutations also changed it. CONCLUSION: Due to their correlation with tissue specificity, CNE2.04-R400 is associated with the tissue-specificity of CNE2.04, and motif CCCCTC plays an important role in the enhancer activity of CNE2.04.


Assuntos
Elementos Facilitadores Genéticos , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Especificidade de Órgãos , Peixe-Zebra/metabolismo
11.
J Vis Exp ; (185)2022 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-35969083

RESUMO

Heart disease is the leading cause of morbidity and mortality worldwide. Due to their low cost, ease of handling, and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.


Assuntos
Arritmias Cardíacas , Transgenes , Animais , Animais Geneticamente Modificados , Arritmias Cardíacas/fisiopatologia , Modelos Animais de Doenças , Coração , Humanos , Camundongos , Miocárdio/metabolismo , Ratos
12.
Artigo em Inglês | MEDLINE | ID: mdl-35940544

RESUMO

Nanomaterials have been widely employed from industrial to medical fields due to their small sizes and versatile characteristics. However, nanomaterials can also induce unexpected adverse effects on health. In particular, exposure of the nervous system to nanomaterials can cause serious neurological dysfunctions and neurodegenerative diseases. A number of studies have adopted various animal models to evaluate the neurotoxic effects of nanomaterials. Among them, zebrafish has become an attractive animal model for neurotoxicological studies due to several advantages, including the well-characterized nervous system, efficient genome editing, convenient generation of transgenic lines, high-resolution in vivo imaging, and an array of behavioral assays. In this review, we summarize recent studies on the neurotoxicological effects of nanomaterials, particularly engineered nanomaterials and nanoplastics, using zebrafish and discuss key findings with advantages and limitations of the zebrafish model in neurotoxicological studies.


Assuntos
Nanoestruturas , Síndromes Neurotóxicas , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Nanoestruturas/toxicidade , Síndromes Neurotóxicas/etiologia , Peixe-Zebra/genética
13.
Methods Mol Biol ; 2540: 35-78, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35980572

RESUMO

Binary expression systems are useful genetic tools for experimentally labeling or manipulating the function of defined cells. The Q-system is a repressible binary expression system that consists of a transcription factor QF (and the recently improved QF2/QF2w), the inhibitor QS, a QUAS-geneX effector, and a drug that inhibits QS (quinic acid). The Q-system can be used alone or in combination with other binary expression systems, such as GAL4/UAS and LexA/LexAop. In this review chapter, we discuss the past, present, and future of the Q-system for applications in Drosophila and other organisms. We discuss the in vivo application of the Q-system for transgenic labeling, the modular nature of QF that allows chimeric or split transcriptional activators to be developed, its temporal control by quinic acid, new methods to generate QF2 reagents, intersectional expression labeling, and its recent adoption into many emerging experimental species.


Assuntos
Proteínas de Drosophila , Ácido Quínico , Animais , Animais Geneticamente Modificados , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ácido Quínico/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transgenes
14.
Methods Mol Biol ; 2540: 113-134, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35980575

RESUMO

Editing the Drosophila genome is incredibly useful for gene functional analysis. However, compared to gene knockouts, precise gene editing is difficult to achieve. Prime editing, a recently described CRISPR/Cas9-based technique, has the potential to make precise editing simpler and faster, and produce less errors than traditional methods. Initially described in mammalian cells, prime editing is functional in Drosophila somatic and germ cells. Here, we outline steps to design, generate, and express prime editing components in transgenic flies. Furthermore, we highlight a crossing scheme to produce edited fly stocks in less than 3 months.


Assuntos
Sistemas CRISPR-Cas , Drosophila , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Drosophila/genética , Edição de Genes/métodos , Genoma de Inseto , Mamíferos/genética
15.
Methods Mol Biol ; 2540: 177-199, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35980578

RESUMO

Overexpression is one of the classical approaches to study pleiotropic functions of genes of interest. To achieve overexpression, we often increase the transcription by introducing genes on exogenous vectors or by using the CRISPR/dCas9-based transcriptional activation system. To date, the most efficient CRISPR/dCas9-based transcriptional activator is the Synergistic Activation Mediator (SAM) system whereby three different transcriptional activation domains are directly fused to dCas9 and MS2 phage Coat Protein (MCP), respectively, and the system in Drosophila is named flySAM. Here we describe the effective and convenient transcriptional activation system, flySAM, starting from vector construction, microinjection, and transgenic fly selection to the phenotypic analysis.


Assuntos
Sistemas CRISPR-Cas , Drosophila , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Drosophila/genética , Drosophila/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional
16.
Methods Mol Biol ; 2540: 349-359, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35980588

RESUMO

Fluorescently labeled transgenic lines of Drosophila melanogaster are a powerful routine tool in fly laboratories. The possibility to fluorescently visualize individual cell populations or entire tissues and the constantly improving microscopy technologies such as two-photon or light-sheet applications, with deep tissue imaging, hold great potential to address central biological questions at an organismic level. However, strong pigmentation and the opaque nature of the D. melanogaster cuticle hinder the penetration of visible light into internal tissues, thereby limiting the application of fluorescent microscopes to analyses of the outermost surfaces of intact samples. In addition, tissue-induced light scattering and optical aberrations quickly blur the view and, hence, require tissue sectioning for further investigation. We have developed a tissue-clearing and depigmentation approach (FlyClear), which preserves endogenous fluorescent signals and is applicable to various developmental stages ranging from larvae to adult fruit flies (Pende et al. Nature communications 9:4731, 2018). In this chapter, we provide a detailed protocol of the experimental steps involved.


Assuntos
Drosophila melanogaster , Drosophila , Animais , Animais Geneticamente Modificados , Imageamento Tridimensional/métodos , Larva , Microscopia de Fluorescência/métodos
17.
Development ; 149(16)2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-35980363

RESUMO

Visualizing cell shapes and interactions of differentiating cells is instrumental for understanding organ development and repair. Across species, strategies for stochastic multicolour labelling have greatly facilitated in vivo cell tracking and mapping neuronal connectivity. Yet integrating multi-fluorophore information into the context of developing zebrafish tissues is challenging given their cytoplasmic localization and spectral incompatibility with common fluorescent markers. Inspired by Drosophila Raeppli, we developed FRaeppli (Fish-Raeppli) by expressing bright membrane- or nuclear-targeted fluorescent proteins for efficient cell shape analysis and tracking. High spatiotemporal activation flexibility is provided by the Gal4/UAS system together with Cre/lox and/or PhiC31 integrase. The distinct spectra of the FRaeppli fluorescent proteins allow simultaneous imaging with GFP and infrared subcellular reporters or tissue landmarks. We demonstrate the suitability of FRaeppli for live imaging of complex internal organs, such as the liver, and have tailored hyperspectral protocols for time-efficient acquisition. Combining FRaeppli with polarity markers revealed previously unknown canalicular topologies between differentiating hepatocytes, reminiscent of the mammalian liver, suggesting common developmental mechanisms. The multispectral FRaeppli toolbox thus enables the comprehensive analysis of intricate cellular morphologies, topologies and lineages at single-cell resolution in zebrafish.


Assuntos
Integrases , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Proteínas de Fluorescência Verde/metabolismo , Integrases/metabolismo , Mamíferos/metabolismo , Neurônios/metabolismo , Peixe-Zebra/metabolismo
18.
Rev Sci Tech ; 41(1): 100-106, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35925632

RESUMO

Insects play a crucial role in research. Many laboratories are developing technologies to control insect vectors or agricultural pests by using genetic modifications that either reduce insect reproduction or increase refractoriness to disease transmission. Those tools include gene-drive elements that may spread such genetic traits in a selfsustaining and cost-effective manner. Since international research collaborations are nowadays routine, movement of genetically modified insects between laboratories under different regulatory jurisdictions is very common. This article describes the requirements and guidelines for transportation of genetically modified insects for research. The author draws upon the experience gained by an Italian laboratory, in its role as a research centre involved in shipments of wild and modified mosquitoes, within an international research consortium developing sustainable tools for malaria control.


Les insectes jouent un rôle essentiel dans la recherche. Nombre de laboratoires mettent actuellement au point des technologies qui visent à lutter contre les insectes vecteurs de maladies ou ravageurs des cultures, en introduisant des modifications génétiques dont le but est de limiter la reproduction des insectes ou de les rendre résistants à la transmission des maladies. Ces outils font appel au guidage génétique, qui permet la transmission héréditaire de ces traits de manière durable et peu onéreuse. Dans le contexte actuel où les collaborations scientifiques au niveau international sont devenues systématiques, les transferts d'insectes génétiquement modifiés entre laboratoires de différents pays sont désormais très fréquents. L'auteur décrit les dispositions réglementaires et les directives applicables au transport d'insectes génétiquement modifiés à des fins de recherche. Il s'appuie sur l'expérience acquise par un laboratoire italien doté du statut de centre de recherche et qui, à ce titre, participe aux expéditions de moustiques sauvages et génétiquement modifiés dans le cadre d'un consortium scientifique international pour la mise au point d'outils durables de lutte contre le paludisme.


Los insectos son un elemento fundamental para la investigación. Numerosos laboratorios están desarrollando técnicas destinadas a combatir a insectos que actúan como vectores o constituyen plagas agrícolas empleando modificaciones genéticas que bien frenan la reproducción del insecto o bien confieren mayor resistencia a la transmisión de enfermedades. Entre tales herramientas figuran elementos de genética dirigida que posibilitan la propagación de esos rasgos genéticos de manera a la vez autosostenida y rentable. Teniendo en cuenta lo habituales que son hoy en día las iniciativas de investigación que entrañan colaboración internacional, el traslado de insectos genéticamente modificados entre laboratorios sujetos a diferentes jurisdicciones reglamentarias es también algo muy común. El autor expone los requisitos y directrices que rigen el transporte de insectos genéticamente modificados con fines de investigación, basándose en la experiencia de un laboratorio italiano que, como parte de un consorcio internacional de investigación que elabora herramientas sostenibles de lucha antipalúdica, cumple funciones de centro de investigación en cuanto al transporte de mosquitos salvajes o modificados.


Assuntos
Culicidae , Malária , Animais , Animais Geneticamente Modificados , Culicidae/genética , Insetos/genética , Laboratórios , Malária/prevenção & controle , Malária/veterinária , Mosquitos Vetores
19.
Dev Cell ; 57(16): 2026-2040.e5, 2022 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-35914525

RESUMO

Cell ablation is a key method in the research fields of developmental biology, tissue regeneration, and tissue homeostasis. Eliminating specific cell populations allows for characterizing interactions that control cell differentiation, death, behavior, and spatial organization of cells. Current methodologies for inducing cell death suffer from relatively slow kinetics, making them unsuitable for analyzing rapid events and following primary and immediate consequences of the ablation. To address this, we developed a cell-ablation system that is based on bacterial toxin/anti-toxin proteins and enables rapid and cell-autonomous elimination of specific cell types and organs in zebrafish embryos. A unique feature of this system is that it uses an anti-toxin, which allows for controlling the degree and timing of ablation and the resulting phenotypes. The transgenic zebrafish generated in this work represent a highly efficient tool for cell ablation, and this approach is applicable to other model organisms as demonstrated here for Drosophila.


Assuntos
Drosophila , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Morte Celular , Diferenciação Celular , Peixe-Zebra/genética
20.
Int J Mol Sci ; 23(16)2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-36012413

RESUMO

The number of reported macrocyclic lactones (ML) resistance cases across all livestock hosts is steadily increasing. Different studies in the parasitic nematode Haemonchus contortus assume the participation of cytochrome P450s (Cyps) enzymes in ML resistance. Still, functional data about their individual contribution to resistance or substrate specificity is missing. Via microinjection, transgenic Caenorhabditis elegans expressing HCON_00141052 (transgene-Hco-cyp-13A11) from extrachromosomal arrays were generated. After 24 h of exposure to different concentrations of ivermectin (IVM), ivermectin aglycone (IVMa), selamectin (SEL), doramectin (DRM), eprinomectin (EPR), and moxidectin (MOX), motility assays were performed to determine the impact of the H. contortus Cyp to the susceptibility of the worms against each ML. While transgene-Hco-cyp-13A11 significantly decreased susceptibility to IVM (four-fold), IVMa (2-fold), and SEL (3-fold), a slight effect for DRM and no effect for MOX, and EPR was observed. This substrate specificity of Hco-cyp-13A11 could not be explained by molecular modeling and docking studies. Hco-Cyp-13A11 molecular models were obtained for alleles from isolates with different resistance statuses. Although 14 amino acid polymorphisms were detected, none was resistance specific. In conclusion, Hco-cyp-13A11 decreased IVM, IVMa, and SEL susceptibility to a different extent, but its potential impact on ML resistance is not driven by polymorphisms.


Assuntos
Anti-Helmínticos , Haemonchus , Animais , Animais Geneticamente Modificados , Anti-Helmínticos/farmacologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Resistência a Medicamentos/genética , Haemonchus/genética , Ivermectina/metabolismo , Ivermectina/farmacologia , Lactonas/metabolismo , Lactonas/farmacologia
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