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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.
J Korean Med Sci ; 34(33): e225, 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31436053

RESUMO

BACKGROUND: Tauopathies, a class of neurodegenerative diseases that includes Alzheimer's disease (AD), are characterized by the deposition of neurofibrillary tangles composed of hyperphosphorylated tau protein in the human brain. As abnormal alterations in histone acetylation and methylation show a cause and effect relationship with AD, we investigated the role of several Jumonji domain-containing histone demethylase (JHDM) genes, which have yet to be studied in AD pathology. METHODS: To examine alterations of several JHDM genes in AD pathology, we performed bioinformatics analyses of JHDM gene expression profiles in brain tissue samples from deceased AD patients. Furthermore, to investigate the possible relationship between alterations in JHDM gene expression profiles and AD pathology in vivo, we examined whether tissue-specific downregulation of JHDM Drosophila homologs (kdm) can affect tauR406W-induced neurotoxicity using transgenic flies containing the UAS-Gal4 binary system. RESULTS: The expression levels of JHDM1A, JHDM2A/2B, and JHDM3A/3B were significantly higher in postmortem brain tissue from patients with AD than from non-demented controls, whereas JHDM1B mRNA levels were downregulated in the brains of patients with AD. Using transgenic flies, we revealed that knockdown of kdm2 (homolog to human JHDM1), kdm3 (homolog to human JHDM2), kdm4a (homolog to human JHDM3A), or kdm4b (homolog to human JHDM3B) genes in the eye ameliorated the tauR406W-engendered defects, resulting in less severe phenotypes. However, kdm4a knockdown in the central nervous system uniquely ameliorated tauR406W-induced locomotion defects by restoring heterochromatin. CONCLUSION: Our results suggest that downregulation of kdm4a expression may be a potential therapeutic target in AD.


Assuntos
Proteínas de Drosophila/genética , Histona Desmetilases/metabolismo , Proteínas tau/genética , Idoso , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Animais Geneticamente Modificados/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Heterocromatina/metabolismo , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/genética , Humanos , Locomoção , Masculino , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Interferência de RNA , Transcriptoma , Proteínas tau/metabolismo
4.
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
5.
Cell Prolif ; 52(5): e12656, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31264309

RESUMO

OBJECTIVES: Cell migration has a key role in cancer metastasis, which contributes to drug resistance and tumour recurrence. Better understanding of the mechanisms involved in this process will potentially reveal new drug targets for cancer therapy. Fer is a non-receptor protein tyrosine kinase aberrantly expressed in various human cancers, whereas its role in tumour progression remains elusive. MATERIALS AND METHODS: Transgenic flies and epigenetic analysis were employed to investigate the role of Drosophila Fer (FER) in cell migration and underlying mechanisms. Co-immunoprecipitation assay was used to monitor the interaction between FER and Drosophila JNK (Bsk). The conservation of Fer in regulating JNK signalling was explored in mammalian cancer and non-cancer cells. RESULTS: Overexpression of FER triggered cell migration and activated JNK signalling in the Drosophila wing disc. Upregulation and downregulation in the basal activity of Bsk exacerbated and eliminated FER-mediated migration, respectively. In addition, loss of FER blocked signal transduction of the JNK pathway. Specifically, FER interacted with and promoted the activity of Bsk, which required both the kinase domain and the C-terminal of Bsk. Lastly, Fer regulated JNK activities in mammalian cells. CONCLUSIONS: Our study reveals FER as a positive regulator of JNK-mediated cell migration and suggests its potential role as a therapeutic target for cancer metastasis.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proteínas de Drosophila/química , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/química , Metaloproteinase 1 da Matriz/metabolismo , Domínios Proteicos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Asas de Animais/metabolismo
6.
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
7.
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
8.
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
9.
Sci Total Environ ; 659: 540-547, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31096383

RESUMO

Mercury is a potentially toxic trace metal that poses threats to aquatic life and to humans. In this study, a mercury-binding peptide was displayed on the surface of Escherichia coli cells using an N-terminal region ice nucleation protein anchor. The surface-engineered E. coli facilitated selective adsorption of mercury ions (Hg2+) from a solution containing various metal ions. The Hg2+ adsorption capacity of the surface-engineered cell was four-fold higher than that of the original E. coli cells. Approximately 95% of Hg2+ was removed from solution by these whole-cell sorbents. The transformed strains were fed to Carassius auratus, so that the bacteria could colonize fish intestine. Engineered bacteria-fed C. auratus showed significantly less (51.1%) accumulation of total mercury when compared with the group that had not been fed engineered bacteria. The surface-engineered E. coli effectively protected fish against the toxicity of Hg2+ in aquatic environments by adsorbing more Hg2+. Furthermore, the surface-engineered E. coli mitigated microbial diversity changes in the intestine caused by Hg2+ exposure, thereby protecting the intestinal microbial community. This strategy is a novel approach for controlling Hg2+ contamination in fish.


Assuntos
Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Carpa Dourada/metabolismo , Mercúrio/metabolismo , Peptídeos/metabolismo , Poluentes Químicos da Água/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Engenharia Genética , Mucosa Intestinal/metabolismo , Microrganismos Geneticamente Modificados/genética , Peptídeos/genética
10.
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
11.
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
12.
PLoS One ; 14(3): e0214257, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30913273

RESUMO

Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codon-optimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOrange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas Luminescentes/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/metabolismo , Autofagia/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/genética , Permeabilidade da Membrana Celular/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Microscopia Confocal , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência
13.
Int J Mol Sci ; 20(3)2019 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-30708986

RESUMO

The creation of functional materials from renewable resources has attracted much interest. We previously reported on the genetic code expansion of the domesticated silkworm Bombyx mori to functionalize silk fiber with synthetic amino acids such as 4-azido-L-phenylalanine (AzPhe). The azido groups act as selective handles for biorthogonal chemical reactions. Here we report the characterization and scaled-up production of azido-functionalized silk fiber for textile, healthcare, and medical applications. To increase the productivity of azido-functionalized silk fiber, the original transgenic line was hybridized with a high silk-producing strain. The F1 hybrid produced circa 1.5 times more silk fibroin than the original transgenic line. The incorporation efficiency of AzPhe into silk fibroin was retained after hybridization. The tensile properties of the azido-functionalized silk fiber were equal to those of normal silk fiber. Scaled-up production of the azido-functionalized silk fiber was demonstrated by rearing circa 1000 transgenic silkworms. Differently-colored fluorescent silk fibers were successfully prepared by click chemistry reactions, demonstrating the utility of the azido-functionalized silk fiber for developing silk-based materials with desired functions.


Assuntos
Azidas/química , Bombyx/genética , Fibroínas/metabolismo , Fenilalanina-tRNA Ligase/genética , Fenilalanina/análogos & derivados , Animais , Animais Geneticamente Modificados/metabolismo , Bombyx/metabolismo , Química Click , Feminino , Fibroínas/química , Código Genético , Proteínas de Insetos/genética , Masculino , Mutação , Fenilalanina/química
14.
Int J Mol Sci ; 20(3)2019 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-30709037

RESUMO

ß-Catenin is an evolutionarily conserved molecule in the canonical Wnt signaling pathway, which controls decisive steps in embryogenesis and functions as a crucial effector in the development of hair follicles. However, the molecular mechanisms underlying wool production have not been fully elucidated. In this study, we investigated the effects of ovine ß-catenin on wool follicles of transgenic sheep produced by pronuclear microinjection with a skin-specific promoter of human keratin14 (k14). Both polymerase chain reaction and Southern blot analysis showed that the sheep carried the ovine ß-catenin gene and that the ß-catenin gene could be stably inherited. To study the molecular responses to high expression of ß-catenin, high-throughput RNA-seq technology was employed using three transgenic sheep and their wild-type siblings. These findings suggest that ß-catenin normally plays an important role in wool follicle development by activating the downstream genes of the Wnt pathway and enhancing the expression of keratin protein genes and keratin-associated protein genes.


Assuntos
Perfilação da Expressão Gênica/veterinária , Queratina-14/genética , Ovinos/genética , Lã/metabolismo , beta Catenina/genética , Animais , Animais Geneticamente Modificados/metabolismo , Feminino , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Microinjeções , Regiões Promotoras Genéticas , Análise de Sequência de RNA , Ovinos/metabolismo , Pele/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo
15.
Transgenic Res ; 28(2): 237-246, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30697646

RESUMO

Producing heterologous enzymes in the animal digestive tract to improve feed utilization rate is a new research strategy by transgenic technology. In this study, transgenic pigs specifically expressing ß-glucanase gene in the intestine were successfully produced by somatic cell nuclear transfer technology in order to improve digestibility of dietary ß-glucan and absorption of nutrients. The ß-glucanase activity in the intestinal juice of 4 transgenic pigs was found to be 8.59 ± 2.49 U/mL. The feeding trial results showed that the crude protein digestion of 4 transgenic pigs was significantly increased compared with that of the non-transgenic pigs. In order to investigate the inheritance of the transgene, 7 G1 transgenic pigs were successfully obtained. The ß-glucanase activity in the intestinal juice of 7 G1 transgenic pigs was found to be 2.35 ± 0.72 U/mL. The feeding trial results showed the crude protein digestion and crude fat digestion were significantly higher in 7 G1 transgenic pigs than in non-transgenic pigs. Taken together, our study demonstrated that the foreign ß-glucanase expressing in the intestine of the transgenic pigs could reduce the anti-nutritional effect of ß-glucans in feed. In addition, ß-glucanase gene could be inherited to the offsprings and maintain its physiological function. It is a promising approach to improve feed utilization by producing transgenic animals.


Assuntos
Ração Animal/análise , Animais Geneticamente Modificados/metabolismo , Glucanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Intestinos/enzimologia , Paenibacillus polymyxa/enzimologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Glicosídeo Hidrolases/genética , Suínos
16.
Transgenic Res ; 28(2): 189-198, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30637610

RESUMO

Xylan is one of the main anti-nutritional factors in pig's feed. Although supplementation of ß-xylanase in diet can improve the utilization of nutrients in animals, it is limited by feed cost, manufacturing process and storage stability. To determine whether the expression of endogenous ß-xylanase gene xynB in vivo can improve digestibility of dietary xylan and absorption of nutrients, we produced transgenic pigs which express the xynB from Aspergillus Niger CGMCC1067 in the parotid gland via nuclear transfer. In four live transgenic founders, ß-xylanase activities in the saliva were 0.74, 0.59, 0.37 and 0.24 U/mL, respectively. Compared with non-transgenic pigs, the content of crude protein (CP) in feces reduced by 15.5% (P < 0.05). Furthermore, in 100 of the 271 F1 pigs the xynB gene was detectable. The digestibility of gross energy and CP in F1 transgenic pigs were increased by 5% and 22%, respectively, with the CP content in feces decreased by 6.4%. Taken together, our study showed that the transgenic pigs producing ß-xylanase from parotid gland can reduce the anti-nutritional effect in animal diet and improve the utilization of nutrients.


Assuntos
Ração Animal/análise , Animais Geneticamente Modificados/metabolismo , Aspergillus niger/enzimologia , Endo-1,4-beta-Xilanases/metabolismo , Nutrientes/análise , Glândula Parótida/metabolismo , Saliva/metabolismo , beta-Glucosidase/metabolismo , Animais , Animais Geneticamente Modificados/genética , Endo-1,4-beta-Xilanases/genética , Suínos , beta-Glucosidase/genética
17.
Methods Mol Biol ; 1854: 13-20, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30027507

RESUMO

Mitochondrial reactive oxygen species (mROS), a major source of ROS within cells, functions as an important signaling molecule and has the ability to damage cellular macromolecules including DNA and proteins. Monitoring mROS levels is therefore essential to understand cell-cell communication and programmed cell death in all types of cell including stem cells. Here, we describe generation and characterization of a redox sensor for mROS that is specifically expressed in the germline stem cells (GSCs) in Drosophila. This redox sensor can be used to monitor the production of mROS and mitophagy in the GSCs during oogenesis.


Assuntos
Drosophila melanogaster/genética , Glutarredoxinas/metabolismo , Mitocôndrias/metabolismo , Células-Tronco de Oogônios/metabolismo , Espécies Reativas de Oxigênio/análise , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Técnicas Biossensoriais , Comunicação Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Feminino , Genes Reporter , Glutarredoxinas/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mitocôndrias/genética , Oxirredução , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/metabolismo
18.
FASEB J ; 33(1): 696-710, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30044923

RESUMO

The proper development of atrioventricular (AV) valves is critical for heart morphogenesis and for the formation of the cardiac conduction system. Defects in AV valve development are the most common type of congenital heart defect. Cardiac troponin I ( ctnni), a structural and regulatory protein involved in cardiac muscle contraction, is a subunit of the troponin complex, but the functions and molecular mechanisms of ctnni during early heart development remain unclear. We created a knockout zebrafish model in which troponin I type 1b ( tnni1b) ( Tnni-HC, heart and craniofacial) was deleted using the clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein system. In the homozygous mutant, the embryos showed severe pericardial edema, malformation of the heart tube, reduction of heart rate without contraction and with almost no blood flow, heart cavity congestion, and lack of an endocardial ring or valve leaflet, resulting in 88.8 ± 6.0% lethality at 7 d postfertilization. Deletion of tnni1b caused the abnormal expression of several markers involved in AV valve development, including bmp4, cspg2, has2, notch1b, spp1, and Alcam. Myocardial re-expression of tnni1b in mutants partially rescued the pericardial edema phenotype and AV canal (AVC) developmental defects. We further showed that tnni1b knockout in zebrafish and ctnni knockdown in rat h9c2 myocardial cells inhibited cardiac wnt signaling and that myocardial reactivation of wnt signaling partially rescued the abnormal expression of AVC markers caused by the tnni1b deletion. Taken together, our data suggest that tnni1b plays a vital role in zebrafish AV valve development by regulating the myocardial wnt signaling pathway.-Cai, C., Sang, C., Du, J., Jia, H., Tu, J., Wan, Q., Bao, B., Xie, S., Huang, Y., Li, A., Li, J., Yang, K., Wang, S., Lu, Q. Knockout of tnni1b in zebrafish causes defects in atrioventricular valve development via the inhibition of myocardial wnt signaling pathway.


Assuntos
Nó Atrioventricular/patologia , Embrião não Mamífero/patologia , Valvas Cardíacas/patologia , Miocárdio/patologia , Troponina I/antagonistas & inibidores , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/antagonistas & inibidores , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Nó Atrioventricular/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Valvas Cardíacas/embriologia , Valvas Cardíacas/metabolismo , Miocárdio/metabolismo , Organogênese , Ratos , Troponina I/genética , Troponina I/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
19.
BMC Biotechnol ; 18(1): 82, 2018 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-30594166

RESUMO

BACKGROUND: The global market for protein drugs has the highest compound annual growth rate of any pharmaceutical class but their availability, especially outside of the US market, is compromised by the high cost of manufacture and validation compared to traditional chemical drugs. Improvements in transgenic technologies allow valuable proteins to be produced by genetically-modified animals; several therapeutic proteins from such animal bioreactors are already on the market after successful clinical trials and regulatory approval. Chickens have lagged behind mammals in bioreactor development, despite a number of potential advantages, due to the historic difficulty in producing transgenic birds, but the production of therapeutic proteins in egg white of transgenic chickens would substantially lower costs across the entire production cycle compared to traditional cell culture-based production systems. This could lead to more affordable treatments and wider markets, including in developing countries and for animal health applications. RESULTS: Here we report the efficient generation of new transgenic chicken lines to optimize protein production in eggs. As proof-of-concept, we describe the expression, purification and functional characterization of three pharmaceutical proteins, the human cytokine interferon α2a and two species-specific Fc fusions of the cytokine CSF1. CONCLUSION: Our work optimizes and validates a transgenic chicken system for the cost-effective production of pure, high quality, biologically active protein for therapeutics and other applications.


Assuntos
Animais Geneticamente Modificados/genética , Biotecnologia/métodos , Galinhas/genética , Citocinas/genética , Animais , Animais Geneticamente Modificados/metabolismo , Reatores Biológicos/economia , Biotecnologia/economia , Galinhas/metabolismo , Citocinas/economia , Citocinas/metabolismo , Humanos , Interferon-alfa/economia , Interferon-alfa/genética , Interferon-alfa/metabolismo , Fator Estimulador de Colônias de Macrófagos/economia , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Proteínas Recombinantes/economia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
Sci Rep ; 8(1): 17710, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30532027

RESUMO

Ecdysteroid UDP glucosyltransferase (EGT) is a baculovirus-encoded protein which can hinder the normal molting of insects by inactivating 20-hydroxyecdysone (20E). Here we expressed EGT in the last-instar silkworm larvae using the GAL4/ UAS system. Compared with the control, for the EGT overexpressed silkworm, the hemolymph 20E content was significantly decreased, the feeding and spinning periods of the last-instar silkworm larvae were extended, the cocoon shell ratio was significantly increased, and the transformation from silkworm larvae to pupa was blocked. Increasing EGT expression resulted in the decrease of 20E content in the hemolymph of silkworm larvae, treating the EGT overexpressed male silkworm with 20E decreased the larval weight and cocoon shell ratio, confirming that the increase in the availability of nutrients to the cocoon and an increase in the cocoon shell weight in the hybrid transgenic silkworms is because of the EGT-induced reduction in active 20E content. Furthermore, though the sericin and flavonoid contents were increased in the cocoon of the EGT overexpressing silkworm, the production of silk fibroin didn't change.


Assuntos
Bombyx/metabolismo , Ecdisteroides/metabolismo , Glucosiltransferases/metabolismo , Larva/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Ecdisterona/metabolismo , Fibroínas/metabolismo , Flavonoides/metabolismo , Masculino , Muda/fisiologia , Pupa/metabolismo , Sericinas/metabolismo
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