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1.
Insect Sci ; 27(1): 14-21, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31246335

RESUMO

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments. The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms. Driven by a given tissue-specific Gal4, expressing UAS-transgene or UAS-RNAi (RNA interference) could be used to up- or down-regulate target gene expression, respectively. However, the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock. Here, we describe a simple way to modulate optomotor blind (omb) expression levels in its endogenous expression region of the wing disc. We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch. The repression effect is more sensitive to temperature than that of overexpression. At low temperature, overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi. In contrast, at high temperature RNAi predominates in gene expression regulation. By this strategy, we could manipulate omb expression levels at a moderate level. It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Proteínas com Domínio T/genética , Asas de Animais/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Proteínas com Domínio T/metabolismo
2.
J Agric Food Chem ; 68(2): 686-696, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31877248

RESUMO

Metabolites of serum and milk from genetically modified (GM) cows and contrast check (CK) cows were comparatively investigated. Serum and milk were collected from genetically modified (GM) cows and contrast check (CK) cows, and then, they were analyzed using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) and gas chromatography-mass spectrometry (GC-MS). Although the level of some blood biochemical indexes for GM cows was shifted up or down, they were generally in normal physiological condition. Serum samples from lactoferrin GM cows exhibited reduced levels of amino acids and elevated levels of indoleacetate, α-keto acids, long-chain fatty acids, etc. GM milk possessed elevated levels of pentose and amino sugar metabolites, including arabitol, xylulose, glucuronate, and N-acetylgalactosamine. Interestingly, some essential nutrients, such as certain unsaturated fatty acids (e.g., eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA)), and some necessary rare sugars were significantly upregulated. Compared to the CK group, a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted based on the increased or decreased metabolites identified in the serum and milk samples of the GM group. The results showed that the GM cows were in healthy condition and their milk has improved benefits for customers. The milk from genetically modified cows was found to be a promising milk source for producing recombinant human lactoferrin (rhLF) for human beings.


Assuntos
Animais Geneticamente Modificados/metabolismo , Lactoferrina/genética , Leite/química , Soro/química , Animais , Animais Geneticamente Modificados/genética , Bovinos/genética , Bovinos/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Feminino , Ácidos Indolacéticos/sangue , Cetoácidos/sangue , Lactoferrina/metabolismo , Metabolômica , Leite/metabolismo , Soro/metabolismo , Açúcares/sangue
3.
Nucleic Acids Res ; 47(19): 10059-10071, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31501873

RESUMO

Delivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the nematode Caenorhabditis elegans, dsRNA from multiple sources can trigger the amplification of silencing signals. Amplification occurs through the production of small RNAs by two RNA-dependent RNA polymerases (RdRPs) that are thought to be tissue-specific - EGO-1 in the germline and RRF-1 in somatic cells. Here we demonstrate that EGO-1 can compensate for the lack of RRF-1 when dsRNA from neurons is used to silence genes in intestinal cells. However, the lineal origins of cells that can use EGO-1 varies. This variability could be because random sets of cells can either receive different amounts of dsRNA from the same source or use different RdRPs to perform the same function. Variability is masked in wild-type animals, which show extensive silencing by neuronal dsRNA. As a result, cells appear similarly functional despite underlying differences that vary from animal to animal. This functional mosaicism cautions against inferring uniformity of mechanism based on uniformity of outcome. We speculate that functional mosaicism could contribute to escape from targeted therapies and could allow developmental systems to drift over evolutionary time.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Inativação Gênica , RNA Replicase/genética , RNA de Cadeia Dupla/genética , Animais , Animais Geneticamente Modificados/genética , Caenorhabditis elegans/genética , Linhagem da Célula/genética , Células Germinativas , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mosaicismo , Neurônios/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética
4.
PLoS Negl Trop Dis ; 13(9): e0007579, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31479450

RESUMO

BACKGROUND: Population suppression through mass-release of Aedes aegypti males carrying dominant-lethal transgenes has been demonstrated in the field. Where population dynamics show negative density-dependence, suppression can be enhanced if lethality occurs after the density-dependent (i.e. larval) stage. Existing molecular tools have limited current examples of such Genetic Pest Management (GPM) systems to achieving this through engineering 'cell-autonomous effectors' i.e. where the expressed deleterious protein is restricted to the cells in which it is expressed-usually under the control of the regulatory elements (e.g. promoter regions) used to build the system. This limits the flexibility of these technologies as regulatory regions with useful spatial, temporal or sex-specific expression patterns may only be employed if the cells they direct expression in are simultaneously sensitive to existing effectors, and also precludes the targeting of extracellular regions such as cell-surface receptors. Expanding the toolset to 'non-cell autonomous' effectors would significantly reduce these limitations. METHODOLOGY/PRINCIPAL FINDINGS: We sought to engineer female-specific, late-acting lethality through employing the Ae. aegypti VitellogeninA1 promoter to drive blood-meal-inducible, fat-body specific expression of tTAV. Initial attempts using pro-apoptotic effectors gave no evident phenotype, potentially due to the lower sensitivity of terminally-differentiated fat-body cells to programmed-death signals. Subsequently, we dissociated the temporal and spatial expression of this system by engineering a novel synthetic effector (Scorpion neurotoxin-TetO-gp67.AaHIT) designed to be secreted out of the tissue in which it was expressed (fat-body) and then affect cells elsewhere (neuro-muscular junctions). This resulted in a striking, temporary-paralysis phenotype after blood-feeding. CONCLUSIONS/SIGNIFICANCE: These results are significant in demonstrating for the first time an engineered 'action at a distance' phenotype in a non-model pest insect. The potential to dissociate temporal and spatial expression patterns of useful endogenous regulatory elements will extend to a variety of other pest insects and effectors.


Assuntos
Aedes/fisiologia , Animais Geneticamente Modificados/fisiologia , Mordeduras e Picadas/parasitologia , Aedes/genética , Animais , Animais Geneticamente Modificados/genética , Mordeduras e Picadas/sangue , Comportamento Alimentar , Feminino , Engenharia Genética , Humanos , Masculino , Controle de Mosquitos , Regiões Promotoras Genéticas , Transgenes
6.
J Sci Food Agric ; 99(15): 6788-6795, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31368537

RESUMO

BACKGROUND: Myostatin (MSTN) negatively regulates skeletal muscle development; however, its functions in internal organs have not been thoroughly investigated. Here, we compared the morphological, molecular, and biological characteristics of the heart, liver, spleen, lungs, kidneys, and tongue of homozygous MSTN mutant (MSTN-/- ), heterozygous MSTN mutant (MSTN+/- ), and wild-type (WT) piglets. RESULTS: The heart and liver were lighter in MSTN-/- piglets than in MSTN+/- piglets, while the tongue was heavier in MSTN-/- piglets than in WT piglets (P < 0.05). Furthermore, the tongue was longer in MSTN-/- piglets than in WT piglets, and myofibers of the tongue were significantly larger in the former piglets than in the latter ones (P < 0.01). mRNA expression of MSTN in all organs was significantly lower in MSTN-/- and MSTN+/- piglets than in WT piglets (P < 0.05). Meanwhile, mRNA expression of follistatin, which is closely related to MSTN, in the heart and liver was significantly higher in MSTN-/- piglets than in MSTN+/- and WT piglets (P < 0.05). In addition, protein expression of MSTN in the heart, kidneys, and tongue was significantly lower in MSTN-/- piglets than in WT piglets (P < 0.01). CONCLUSION: These results suggest that MSTN is widely expressed and has marked effects in multiple internal organs. Myostatin has crucial functions in regulating internal organ size, especially the tongue. © 2019 Society of Chemical Industry.


Assuntos
Estruturas Animais/crescimento & desenvolvimento , Animais Geneticamente Modificados/crescimento & desenvolvimento , Miostatina/genética , Suínos/crescimento & desenvolvimento , Suínos/genética , Estruturas Animais/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Folistatina/genética , Folistatina/metabolismo , Mutação , Miostatina/metabolismo , Tamanho do Órgão , Suínos/metabolismo
7.
Transgenic Res ; 28(Suppl 2): 45-52, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31321682

RESUMO

Genome editing, particularly using of site-directed nucleases such as the CRISPR system, has spread rapidly through the biological sciences. Genome editing in crops could significantly speed up the progress of breeding programs. It could drive the development of traits in new crops and allow improvements in yield and pest resistance, adaptation to climate change, and industrial and pharmaceutical applications. However biofortification is a key challenge to satisfy nutritional needs in vitamins for developing countries and new consumer's needs for developed countries. China and the USA lead scientific research in crop editing. Nigeria, being headquarters to numerous research consortia, is the most involved country in Africa. Genome editing in animals including pig, cattle, sheep, and carp, has not merely accelerated research but has made possible research that was previously unfeasible. It has been used to increase disease resistance, to make livestock better adapted to farming or environmental conditions, to increase fertility and growth, and to improve animal welfare. The USA, the UK and China are the most involved countries in animal genome editing. Global food production needs to increase as much as 70 per cent to support the growing population. Genome editing could contribute improving the efficiency of food distribution and reducing waste. Depending on the regulatory conditions, genome editing could open up the field to smaller companies and public labs.


Assuntos
Agricultura/tendências , Animais Geneticamente Modificados/genética , Edição de Genes/métodos , Plantas Geneticamente Modificadas/genética , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Cruzamento , Carpas/genética , Bovinos , China , Produtos Agrícolas , Resistência à Doença , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Suínos/genética , Reino Unido , Estados Unidos
8.
PLoS Genet ; 15(6): e1008158, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194738

RESUMO

With the approach of winter, many insects switch to an alternative protective developmental program called diapause. Drosophila melanogaster females overwinter as adults by inducing a reproductive arrest that is characterized by inhibition of ovarian development at previtellogenic stages. The insulin producing cells (IPCs) are key regulators of this process, since they produce and release insulin-like peptides that act as diapause-antagonizing hormones. Here we show that in D. melanogaster two neuropeptides, Pigment Dispersing Factor (PDF) and short Neuropeptide F (sNPF) inhibit reproductive arrest, likely through modulation of the IPCs. In particular, genetic manipulations of the PDF-expressing neurons, which include the sNPF-producing small ventral Lateral Neurons (s-LNvs), modulated the levels of reproductive dormancy, suggesting the involvement of both neuropeptides. We expressed a genetically encoded cAMP sensor in the IPCs and challenged brain explants with synthetic PDF and sNPF. Bath applications of both neuropeptides increased cAMP levels in the IPCs, even more so when they were applied together, suggesting a synergistic effect. Bath application of sNPF additionally increased Ca2+ levels in the IPCs. Our results indicate that PDF and sNPF inhibit reproductive dormancy by maintaining the IPCs in an active state.


Assuntos
Proteínas CLOCK/genética , Proteínas de Drosophila/genética , Neuropeptídeos/genética , Reprodução/genética , Animais , Animais Geneticamente Modificados/genética , Encéfalo/metabolismo , Ritmo Circadiano/genética , Diapausa/genética , Diapausa/fisiologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica/genética , Insulina/genética , Neurônios/metabolismo , Transdução de Sinais/genética
9.
PLoS Genet ; 15(6): e1008221, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31242186

RESUMO

Wolbachia are maternally inherited bacteria that infect arthropod species worldwide and are deployed in vector control to curb arboviral spread using cytoplasmic incompatibility (CI). CI kills embryos when an infected male mates with an uninfected female, but the lethality is rescued if the female and her embryos are likewise infected. Two phage WO genes, cifAwMel and cifBwMel from the wMel Wolbachia deployed in vector control, transgenically recapitulate variably penetrant CI, and one of the same genes, cifAwMel, rescues wild type CI. The proposed Two-by-One genetic model predicts that CI and rescue can be recapitulated by transgenic expression alone and that dual cifAwMel and cifBwMel expression can recapitulate strong CI. Here, we use hatch rate and gene expression analyses in transgenic Drosophila melanogaster to demonstrate that CI and rescue can be synthetically recapitulated in full, and strong, transgenic CI comparable to wild type CI is achievable. These data explicitly validate the Two-by-One model in wMel-infected D. melanogaster, establish a robust system for transgenic studies of CI in a model system, and represent the first case of completely engineering male and female animal reproduction to depend upon bacteriophage gene products.


Assuntos
Bacteriófagos/genética , Drosophila melanogaster/genética , Proteínas Virais/genética , Wolbachia/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/microbiologia , Vetores de Doenças , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/microbiologia , Feminino , Regulação da Expressão Gênica/genética , Masculino , Herança Materna/genética , Reprodução/genética , Wolbachia/patogenicidade , Wolbachia/virologia
10.
Science ; 364(6443): 894-897, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-31147521

RESUMO

Malaria control efforts require implementation of new technologies that manage insecticide resistance. Metarhizium pingshaense provides an effective, mosquito-specific delivery system for potent insect-selective toxins. A semifield trial in a MosquitoSphere (a contained, near-natural environment) in Soumousso, a region of Burkina Faso where malaria is endemic, confirmed that the expression of an insect-specific toxin (Hybrid) increased fungal lethality and the likelihood that insecticide-resistant mosquitoes would be eliminated from a site. Also, as Hybrid-expressing M. pingshaense is effective at very low spore doses, its efficacy lasted longer than that of the unmodified Metarhizium Deployment of transgenic Metarhizium against mosquitoes could (subject to appropriate registration) be rapid, with products that could synergistically integrate with existing chemical control strategies to avert insecticide resistance.


Assuntos
Culicidae/microbiologia , Malária/prevenção & controle , Metarhizium/genética , Controle de Mosquitos/métodos , Venenos de Aranha/genética , Animais , Animais Geneticamente Modificados/genética , Burkina Faso/epidemiologia , Resistência a Inseticidas , Transgenes
11.
Int J Dev Biol ; 63(6-7): 259-270, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250909

RESUMO

Mechanisms of programmed cell death differ between animals, plants and fungi. In animals, apoptotic cell death depends on caspases and Bcl-2 family proteins. These protein families are only found in multicellular animals, including cnidarians, insects and mammals. In contrast, members of the TMBIM-family of transmembrane proteins are conserved across all eukaryotes. Sequence comparisons of cell death related proteins between phyla indicate strong conservation of the genes involved. However, often it is not known whether this is paralleled by conservation of function. Here we present the first study to support an anti-apoptotic function of Bcl-2 like proteins in the cnidarian Hydra within a physiological context. We used transgenic Hydra expressing GFP-tagged HyBcl-2-like 4 protein in epithelial cells. The protein was localised to mitochondria and able to protect Hydra epithelial cells from apoptosis induced by either the PI(3) kinase inhibitor wortmannin or by starvation. Moreover, we identified members of the TMBIM-family in Hydra including HyBax-Inhibitor-1, HyLifeguard-1a and -1b and HyLifeguard 4. Expressing these TMBIM-family members in Hydra and human HEK cells, we found HyBax-inhibitor-1 protein localised to ER-membranes and HyLifeguard-family members localised to the plasma membrane and Golgi-vesicles. Moreover, HyBax-inhibitor-1 protected human cells from camptothecin induced apoptosis. This work illustrates that the investigated Bcl-2- and TMBIM-family members represent evolutionarily conserved mitochondrial, ER, Golgi and plasma membrane proteins with anti-apoptotic functions. The participation of ER and Golgi proteins in the regulation of programmed cell death might be a very ancient feature.


Assuntos
Animais Geneticamente Modificados/metabolismo , Apoptose , Regulação da Expressão Gênica , Hydra/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Células HEK293 , Humanos , Hydra/efeitos dos fármacos , Hydra/genética , Imunossupressores/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Homologia de Sequência , Inanição , Wortmanina/farmacologia , Proteína X Associada a bcl-2/genética
12.
Int J Dev Biol ; 63(6-7): 281-286, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250911

RESUMO

Urodele amphibian newts have an outstanding history as experimental animals in various research fields such as developmental biology and regeneration biology. We have reported a model experimental system using the Spanish newt, Pleurodeles waltl, and it enables reverse/molecular genetics through gene manipulation. Microinjection is one of the core techniques in gene manipulation in newts. In the present study, we examined the conditions of the microinjection method, such as egg preparation, de-jelly solution, and formulation of injection medium. We have successfully optimized the injection protocol for P. waltl newts, and our improved protocol is more efficient and lower in cost than previous methods. This protocol can be used for the microinjection of plasmid DNA with I-SceI or mRNA, as well as genome editing using the CRISPR-Cas9 system. This protocol will facilitate research through gene manipulation in newts.


Assuntos
Animais Geneticamente Modificados/genética , Técnicas de Transferência de Genes , Microinjeções/métodos , Óvulo/metabolismo , Pleurodeles/genética , RNA Mensageiro/administração & dosagem , Espermatozoides/metabolismo , Animais , Animais Geneticamente Modificados/fisiologia , Masculino , Óvulo/crescimento & desenvolvimento , Plasmídeos/administração & dosagem , Plasmídeos/genética , Pleurodeles/fisiologia , RNA Mensageiro/genética , Regeneração , Espermatozoides/crescimento & desenvolvimento
13.
Mol Genet Genomics ; 294(6): 1375-1383, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31214765

RESUMO

Forkhead box O (FoxO) is a downstream transcription factor of the insulin-signaling pathway, which plays vital roles in the growth and metabolism of organisms. In this study, BmFoxO was overexpressed in BmE cells, in which proliferation was inhibited and apoptosis was increased. The transgenic vector overexpressing BmFoxO was constructed, and the transgenic silkworm line A4FoxO was generated via embryonic microinjection. The body size of A4FoxO silkworm was smaller than that of non-transgenic silkworm (WT). The quantitative polymerase chain reaction results revealed that the insulin pathway was enhanced and the growth-related TOR pathway was suppressed. Furthermore, the translation of proteins in the fat body of A4FoxO silkworm was inhibited. The expression level of genes involved in the glucose synthesis and lipolysis pathways was increased, whereas that of genes involved in fat synthesis was decreased. Oil red O staining revealed that the amount of lipid droplets was reduced in A4FoxO silkworms compared with WT. Further analysis showed that the content of triglyceride and glycogen was significantly decreased in fat body, but the content of glucose and trehalose was increased in the hemolymph of A4FoxO silkworms. These results suggest that the enhanced expression of BmFoxO disturbs glycolipid metabolism and affects silkworm growth.


Assuntos
Bombyx/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Glucose/biossíntese , Proteínas de Insetos/metabolismo , Lipólise , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Bombyx/embriologia , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Linhagem Celular , Proliferação de Células , Corpo Adiposo/metabolismo , Fatores de Transcrição Forkhead/genética , Proteínas de Insetos/genética , Larva/crescimento & desenvolvimento , Metabolismo dos Lipídeos/genética , Lipólise/genética , Açúcares/metabolismo , Triglicerídeos/metabolismo
14.
BMC Genomics ; 20(1): 336, 2019 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053056

RESUMO

BACKGROUND: Triploid coho salmon are excellent models for studying gene dosage and the effects of increased cell volume on gene expression. Triploids have an additional haploid genome in each cell and have fewer but larger cells than diploid coho salmon to accommodate the increased genome size. Studying gene expression in triploid coho salmon provides insight into how gene expression may have been affected after the salmonid-specific genome duplication which occurred some 90 MYA. Triploid coho salmon are sterile and consequently can live longer and grow larger than diploid congeners in many semelparous species (spawning only once) because they never reach maturity and post-spawning mortality is averted. Triploid fishes are also of interest to the commercial sector (larger fish are more valuable) and to fisheries management since sterile fish can potentially minimize negative impacts of escaped fish in the wild. RESULTS: The vast majority of genes in liver tissue had similar expression levels between diploid and triploid coho salmon, indicating that the same amount of mRNA transcripts were being produced per gene copy (positive gene dosage effects) within a larger volume cell. Several genes related to nutrition and compensatory growth were differentially expressed between diploid and triploid salmon, indicating that some loci are sensitive to cell size and/or DNA content per cell. To examine how robust expression between ploidies is under different conditions, a genetic/metabolic modifier in the form of different doses of a growth hormone transgene was used to assess gene expression under conditions that the genome has not naturally experienced or adapted to. While many (up to 1400) genes were differentially expressed between non-transgenic and transgenic fish, relatively few genes were differentially expressed between diploids and triploids with similar doses of the transgene. These observations indicate that the small effect of ploidy on gene expression is robust to large changes in physiological state. CONCLUSIONS: These findings are of interest from a gene regulatory perspective, but also valuable for understanding phenotypic effects in triploids, transgenics, and triploid transgenics that could affect their utility in culture conditions and their fitness and potential consequences of release into nature.


Assuntos
Animais Geneticamente Modificados/genética , Diploide , Regulação da Expressão Gênica , Hormônio do Crescimento/administração & dosagem , Fígado/metabolismo , Oncorhynchus kisutch/genética , Triploidia , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Hormônio do Crescimento/genética , Oncorhynchus kisutch/crescimento & desenvolvimento , Oncorhynchus kisutch/metabolismo , Transgenes
15.
Nat Commun ; 10(1): 2113, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068592

RESUMO

Gene editing by CRISPR/Cas9 is commonly used to generate germline mutations or perform in vitro screens, but applicability for in vivo screening has so far been limited. Recently, it was shown that in Drosophila, Cas9 expression could be limited to a desired group of cells, allowing tissue-specific mutagenesis. Here, we thoroughly characterize tissue-specific (ts)CRISPR within the complex neuronal system of the Drosophila mushroom body. We report the generation of a library of gRNA-expressing plasmids and fly lines using optimized tools, which provides a valuable resource to the fly community. We demonstrate the application of our library in a large-scale in vivo screen, which reveals insights into developmental neuronal remodeling.


Assuntos
Animais Geneticamente Modificados/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Drosophila/genética , Edição de Genes/métodos , Animais , Sistemas CRISPR-Cas/genética , Feminino , Masculino , Corpos Pedunculados/metabolismo , Mutagênese , Sistema Nervoso/crescimento & desenvolvimento , Plasticidade Neuronal/genética , Neurônios/fisiologia , Plasmídeos/genética , RNA Guia/genética
16.
Genes Cells ; 24(7): 496-510, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31124270

RESUMO

In the Drosophila brain, neurons form genetically specified synaptic connections with defined neuronal targets. It is proposed that each central nervous system neuron expresses specific cell surface proteins, which act as identification tags. Through an RNAi screen of cell surface molecules in the Drosophila visual system, we found that the cell adhesion molecule Klingon (Klg) plays an important role in repressing the ectopic formation of extended axons, preventing the formation of excessive synapses. Cell-specific manipulation of klg showed that Klg is required in both photoreceptors and the glia, suggesting that the balanced homophilic interaction between photoreceptor axons and the glia is required for normal synapse formation. Previous studies suggested that Klg binds to cDIP and our genetic analyses indicate that cDIP is required in glia for ectopic synaptic repression. These data suggest that Klg play a critical role together with cDIP in refining synaptic specificity and preventing unnecessary connections in the brain.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Proteínas do Olho/metabolismo , Neuroglia/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Sinapses/fisiologia , Vias Visuais , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/fisiologia , Axônios/fisiologia , Moléculas de Adesão Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas do Olho/genética , Feminino
17.
BMC Biol ; 17(1): 34, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30995910

RESUMO

BACKGROUND: Ionotropic receptors (IRs) are a large, divergent subfamily of ionotropic glutamate receptors (iGluRs) that are expressed in diverse peripheral sensory neurons and function in olfaction, taste, hygrosensation and thermosensation. Analogous to the cell biological properties of their synaptic iGluR ancestors, IRs are thought to form heteromeric complexes that localise to the ciliated dendrites of sensory neurons. IR complexes are composed of selectively expressed 'tuning' receptors and one of two broadly expressed co-receptors (IR8a or IR25a). While the extracellular ligand-binding domain (LBD) of tuning IRs is likely to define the stimulus specificity of the complex, the role of this domain in co-receptors is unclear. RESULTS: We identify a sequence in the co-receptor LBD, the 'co-receptor extra loop' (CREL), which is conserved across IR8a and IR25a orthologues but not present in either tuning IRs or iGluRs. The CREL contains a single predicted N-glycosylation site, which we show bears a sugar modification in recombinantly expressed IR8a. Using the Drosophila olfactory system as an in vivo model, we find that a transgenically encoded IR8a mutant in which the CREL cannot be N-glycosylated is impaired in localisation to cilia in some, though not all, populations of sensory neurons expressing different tuning IRs. This defect can be complemented by the presence of endogenous wild-type IR8a, indicating that IR complexes contain at least two IR8a subunits and that this post-translational modification is dispensable for protein folding or complex assembly. Analysis of the subcellular distribution of the mutant protein suggests that its absence from sensory cilia is due to a failure in exit from the endoplasmic reticulum. Protein modelling and in vivo analysis of tuning IR and co-receptor subunit interactions by a fluorescent protein fragment complementation assay reveal that the CREL N-glycosylation site is likely to be located on the external face of a heterotetrameric IR complex. CONCLUSIONS: Our data reveal an important role for the IR co-receptor LBD in control of intracellular transport, provide novel insights into the stoichiometry and assembly of IR complexes and uncover an unexpected heterogeneity in the trafficking regulation of this sensory receptor family.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Receptores Ionotrópicos de Glutamato/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Transporte Proteico , Receptores Ionotrópicos de Glutamato/química , Receptores Ionotrópicos de Glutamato/metabolismo , Alinhamento de Sequência
18.
Cells ; 8(4)2019 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-31027317

RESUMO

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


Assuntos
Engenharia Genética/métodos , Queratina-19/metabolismo , Osteopontina/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Ductos Biliares/metabolismo , Linhagem da Célula/efeitos dos fármacos , Feminino , Expressão Gênica/genética , Expressão Gênica/fisiologia , Integrases/biossíntese , Integrases/genética , Integrases/metabolismo , Queratina-19/genética , Queratina-19/fisiologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos/genética , Osteopontina/genética , Osteopontina/fisiologia , Proteínas Recombinantes/metabolismo , Tamoxifeno/farmacologia
19.
Nat Commun ; 10(1): 1640, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967548

RESUMO

Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. Here, we generalize this "active genetic" approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. We test two configurations for allelic-drive: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located in close proximity to the gRNA cut site. We also characterize a phenomenon we refer to as lethal-mosaicism that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process we refer to as "shadow-drive", have broad practical applications in improving health and agriculture and greatly extend the active genetics toolbox.


Assuntos
Alelos , Reparo do DNA por Junção de Extremidades/genética , Drosophila/genética , Tecnologia de Impulso Genético/métodos , Agricultura/métodos , Animais , Animais Geneticamente Modificados/genética , Sistemas CRISPR-Cas/genética , Análise Mutacional de DNA , Feminino , Edição de Genes/métodos , Padrões de Herança/genética , Masculino , Mosaicismo , RNA Guia/genética
20.
PLoS Genet ; 15(3): e1008002, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30893315

RESUMO

Mammary epithelial progenitors are the normal cell-of-origin of breast cancer. We previously defined a population of p27+ quiescent hormone-responsive progenitor cells in the normal human breast whose frequency associates with breast cancer risk. Here, we describe that deletion of the Cdkn1b gene encoding the p27 cyclin-dependent kinase inhibitor in the estrogen-induced mammary tumor-susceptible ACI rat strain leads to a decrease in the relative frequencies of Cd49b+ mammary luminal epithelial progenitors and pregnancy-related differentiation. We show by comprehensive gene expression profiling of purified progenitor and differentiated mammary epithelial cell populations that p27 deletion has the most pronounced effects on luminal progenitors. Cdkn1b-/- females have decreased fertility, but rats that are able to get pregnant had normal litter size and were able to nurse their pups implying that loss of p27 in ACI rats does not completely abrogate ovarian function and lactation. Reciprocal mammary gland transplantation experiments indicate that the p27-loss-induced changes in mammary epithelial cells are not only caused by alterations in their intrinsic properties, but are likely due to altered hormonal signaling triggered by the perturbed systemic endocrine environment observed in Cdkn1b-/- females. We also observed a decrease in the frequency of mammary epithelial cells positive for progesterone receptor (Pr) and FoxA1, known direct transcriptional targets of the estrogen receptor (Erα), and an increase in phospho-Stat5 positive cells commonly induced by prolactin (Prl). Characterization of genome-wide Pr chromatin binding revealed distinct binding patterns in mammary epithelial cells of Cdkn1b+/+ and Cdkn1b-/- females and enrichment in genes with known roles in Notch, ErbB, leptin, and Erα signaling and regulation of G1-S transition. Our data support a role for p27 in regulating the pool size of hormone-responsive luminal progenitors that could impact breast cancer risk.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Animais , Animais Geneticamente Modificados/genética , Neoplasias da Mama/genética , Diferenciação Celular , Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Endócrinas/fisiologia , Células Epiteliais , Receptor alfa de Estrogênio , Estrogênios , Feminino , Predisposição Genética para Doença/genética , Humanos , Integrina alfa1 , Glândulas Mamárias Animais , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Gravidez , Progesterona , Ratos , Ratos Endogâmicos ACI , Ratos Sprague-Dawley , Receptores Estrogênicos , Receptores de Progesterona , Fatores de Risco , Transdução de Sinais , Células-Tronco
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