RESUMO
As in other vertebrates, fish reproduction is tightly controlled by gonadotropin signaling. One of the most perplexing aspects of gonadotropin action on germ cell biology is the restricted expression of gonadotropin receptors in somatic cells of the gonads. Therefore, the identification of factors conveying the action of gonadotropins on germ cells is particularly important for understanding the mechanism of reproduction. Insulin-like growth factors (Igfs) are recognized as key factors in regulating reproduction by triggering a series of physiological processes in vertebrates. Recently, a novel member of Igfs called Igf3 has been identified in teleost. Different from the conventional Igf1 and Igf2 that are ubiquitously expressed in a majority of tissues, Igf3 is solely or highly expressed in the fish gonads. The role of Igf3 in mediating the action of gonadotropin through Igf type 1 receptor on several aspects of oogenesis and spermatogenesis have been demonstrated in several fish species. In this review, we will summarize existing data on Igf3. This new information obtained from Igf3 provides insight into elucidating the molecular mechanism of fish reproduction, and also highlights the importance of Igf system in mediating the action of gonadotropin signaling on animal reproduction.
Assuntos
Gônadas , Reprodução , Animais , Peixes , Masculino , Oogênese , EspermatogêneseRESUMO
Zebrafish gonadal sexual differentiation is an important but poorly understood subject. Previously, we have identified a novel insulin-like growth factor (Igf) named insulin-like growth factor 3 (Igf3) in teleosts. The importance of Igf3 in oocyte maturation and ovulation has been recently demonstrated by us in zebrafish. In this study, we have further found the essential role of Igf3 in gonadal sexual differentiation of zebrafish. A differential expression pattern of igf3 between ovary and testis during sex differentiation (higher level in ovary than in testis) was found in zebrafish. An igf3 knockout zebrafish line was established using TALENs-mediated gene knockout technique. Intriguingly, all igf3 homozygous mutants were males due to the female-to-male sex reversal occurred during sex differentiation. Further analysis showed that Igf3 did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells. Oocyte development was arrested at primary growth stage, and the ovary was gradually sex-reversed to testis before 60 day post fertilization (dpf). Such sex reversal was likely due to decreased germ cell proliferation by suppressing PI3K/Akt pathway in early ovaries of igf3 mutants. Estrogen is considered as a master regulator in fish sex differentiation. Here, we found that igf3 expression could be upregulated by estrogen in early stages of ovarian follicles as evidenced in in vitro treatment assays and cyp19a1a mutant zebrafish, and E2 failed to rescue the defects of igf3 mutants in ovarian development, suggesting that Igf3 may serve as a downstream factor of estrogen signaling in sex differentiation. Taken together, we demonstrated that Igf3 is essential for ovary differentiation in zebrafish.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Ovário/crescimento & desenvolvimento , Diferenciação Sexual/fisiologia , Somatomedinas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Apoptose , Proliferação de Células , Feminino , Masculino , Mutação , Oócitos/crescimento & desenvolvimento , Somatomedinas/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
A novel insulin-like growth factor (igf3), which is exclusively expressed in the gonads, has been widely identified in fish species. Recent studies have indicated that Igf3 regulates spermatogonia proliferation and differentiation in zebrafish; however, detailed information on the role of this Igf needs further in vivo investigation. Here, using Nile tilapia (Oreochromis niloticus) as an animal model, we report that igf3 is required for spermatogenesis and reproduction. Knockout of igf3 by CRISPR/Cas9 severely inhibited spermatogonial proliferation and differentiation at 90 days after hatching, the time critical for meiosis initiation, and resulted in less spermatocytes in the mutants. Although spermatogenesis continued to occur later, more spermatocytes and less spermatids were observed in the igf3-/- testes when compared with wild type of testes at adults, indicating that Igf3 regulates spermatocyte to spermatid transition. Importantly, a significantly increased occurrence of apoptosis in spermatids was observed after loss of Igf3. Therefore, igf3-/- males were subfertile with drastically reduced semen volume and sperm count. Conversely, the overexpression of Igf3 in XY tilapia enhanced spermatogenesis leading to more spermatids and sperm count. Transcriptomic analysis revealed that the absence of Igf3 resulted in dysregulation of many genes involved in cell cycle, meiosis and pluripotency regulators that are critical for spermatogenesis. In addition, in vitro gonadal culture with 17α-methyltetosterone (MT) and 11-ketotestosterone (11-KT) administration and in vivo knockout of cyp11c1 demonstrated that igf3 expression is regulated by androgens, suggesting that Igf3 acts downstream of androgens in fish spermatogenesis. Notably, the igf3 knockout did not affect body growth, indicating that this Igf specifically functions in reproduction. Taken together, our data provide genetic evidence for fish igf3 in the regulation of reproductive capacity by controlling spermatogenesis.
Assuntos
Reprodução , Somatomedinas/metabolismo , Espermatogênese , Tilápia/fisiologia , Androgênios/farmacologia , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Masculino , Camundongos Knockout , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodução/efeitos dos fármacos , Reprodução/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Somatomedinas/genética , Espermatogênese/efeitos dos fármacos , Espermatogênese/genética , Espermatogônias/citologia , Espermatogônias/efeitos dos fármacos , Espermatogônias/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismo , Tilápia/genéticaRESUMO
Thraustochytriidae sp. have broadly gained attention as a prospective resource for the production of omega-3 fatty acids production in significant quantities. In this study, the whole genome of Thraustochytriidae sp. SZU445, which produces high levels of docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), was sequenced and subjected to protein annotation. The obtained clean reads (63.55 Mb in total) were assembled into 54 contigs and 25 scaffolds, with maximum and minimum lengths of 400 and 0.0054 Mb, respectively. A total of 3513 genes (24.84%) were identified, which could be classified into six pathways and 44 pathway groups, of which 68 genes (1.93%) were involved in lipid metabolism. In the Gene Ontology database, 22,436 genes were annotated as cellular component (8579 genes, 38.24%), molecular function (5236 genes, 23.34%), and biological process (8621 genes, 38.42%). Four enzymes corresponding to the classic fatty acid synthase (FAS) pathway and three enzymes corresponding to the classic polyketide synthase (PKS) pathway were identified in Thraustochytriidae sp. SZU445. Although PKS pathway-associated dehydratase and isomerase enzymes were not detected in Thraustochytriidae sp. SZU445, a putative DHA- and DPA-specific fatty acid pathway was identified.
Assuntos
Ácidos Graxos Insaturados/biossíntese , Ácidos Graxos Insaturados/genética , Estramenópilas/genética , Proteínas de Algas/genética , Ácidos Docosa-Hexaenoicos , Ácido Graxo Sintases/genética , Ontologia Genética , Policetídeo Sintases/genética , Estudos Prospectivos , Análise de Sequência de DNARESUMO
The role of androgenic steroids on ovarian development has attracted much attention in recent years, but the molecular mechanism is still largely unknown. In this study, using zebrafish as a model, we found that the trace metal zinc mediates the action of androgen on oocyte maturation. The ovarian and serum testosterone is transiently stimulated by LH during oocyte maturation. Testosterone could mimic the action of LH on oocyte maturation, and its action appears to be independent of the classical nuclear androgen receptor. Consistent with a recent finding that a zinc transporter (Zip9) has been suggested as a novel androgen receptor, we found the labile zinc concentration could be induced by testosterone in the ovarian follicular cells, and zinc could mimic the action of testosterone on oocyte maturation and signaling. Moreover, the action of testosterone on oocyte maturation could be abolished by the chelation of zinc. Thus, the evidence supports the notion that zinc could mediate the action of androgen on oocyte maturation in zebrafish. This finding would shed light on understanding the role of androgen in ovary development and the molecular mechanism of oocyte maturation in fish.
Assuntos
Androgênios/metabolismo , Hormônio Luteinizante/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Zinco/farmacologia , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio Luteinizante/genética , Oogênese/efeitos dos fármacos , Receptores Androgênicos , Testosterona/farmacologia , Peixe-ZebraRESUMO
BACKGROUND: Prognostication of patients with colorectal cancer (CRC) currently relies on tumor-node-metastasis (TNM) staging but clinical outcomes of patients of the same histoclinical stage are heterogeneous. It is therefore imperative to devise novel molecular tests to stratify CRC patients. Our previous work demonstrated that eukaryotic elongation factor-2 kinase (EEF2K) is a tumor suppressor in CRC. Herein, we investigated EEF2K expression in CRC and determined its relationship with clinicopathological parameters. METHODS: Quantitative RT-PCR and Westerns blots were used to examine EEF2K expression in primary tumor and the adjacent non-tumor tissues of CRC patients (n = 20). Kaplan-Meier curves and Cox regression analysis were used to assess the association between clinical outcomes of CRC patients and EEF2K protein expression determined by immunohistochemistry on tissue microarray (n = 151). RESULTS: EEF2K was significantly downregulated at both mRNA and protein levels in tumors of CRC patients. Univariate Cox regression analysis revealed that CRC patients with high tumor grade, advanced TNM staging and low EEF2K expression were associated with worse overall survival. Multivariate analysis further demonstrated that low EEF2K expression was an independent factor for predicting poorer overall survival in CRC patients (p = 0.014; Hazard ratio = 2.951; 95% confidence interval: 1.240-7.024). The 5-year survival rate was 82.8% in the EEF2K-high-expression group versus 63.9% in the EEF2K-low-expression group (p = 0.0118). The association of overall survival with EEF2K expression in CRC patients was verified in The Cancer Genome Atlas (TCGA) cohort. CONCLUSIONS: EEF2K is downregulated in CRC and its expression can be employed as a prognostic marker for CRC patients independent of TNM staging.
Assuntos
Neoplasias do Colo/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Neoplasias Retais/metabolismo , Idoso , Colo/metabolismo , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Regulação para Baixo , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/metabolismo , Neoplasias Retais/mortalidade , Neoplasias Retais/patologia , Reto/metabolismo , Análise de Regressão , Taxa de Sobrevida , Resultado do Tratamento , Proteínas Supressoras de TumorRESUMO
Gonadal development is precisely regulated by the two gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Much progress on understanding the functions of LH and FSH signaling on gonad development has been achieved in the past decades, mostly from studies in mammals, especially genetic studies in both mouse and human. The functions of both LH and FSH signaling in nonmammalian species are still largely unknown. In recent years, using zebrafish, a teleost phylogenetically distant from mammals, we and others have genetically analyzed the functions of gonadotropins and their receptors through gene knockout studies. In this review, we will summarize the pertinent findings and discuss how the actions of gonadotropin signaling on gonad development have evolved during evolution from fish to mammals.
Assuntos
Gonadotropinas/fisiologia , Gônadas/crescimento & desenvolvimento , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Evolução Molecular , Feminino , Técnicas de Inativação de Genes , Gonadotropinas/deficiência , Gonadotropinas/genética , Gônadas/fisiologia , Masculino , Ovário/crescimento & desenvolvimento , Ovário/fisiologia , Filogenia , Nós Neurofibrosos , Receptores da Gonadotropina/deficiência , Receptores da Gonadotropina/genética , Receptores da Gonadotropina/fisiologia , Transdução de Sinais , Testículo/crescimento & desenvolvimento , Testículo/fisiologia , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Both oocyte maturation and ovulation is triggered by the luteinizing hormone (LH) surge in vertebrates, but exactly how these processes are regulated by LH remains to be fully elucidated. Previously, we found that Igf3, a fish-specific member of the igf family predominantly expressed in the gonads, could mediate the action of LH on oocyte maturation in zebrafish. Here, we further reveal the importance of Igf3 in mediating the action of LH on ovulation in zebrafish. All the four igf gene family members are expressed in the zebrafish ovary but only the igf3 transcript level is increased in hCG-induced ovulation in vivo. The expression of Igf3 protein in the follicles is also increased during ovulation. The actions of hCG on the expression of ovulatory enzymes and on ovulation itself could be largely mimicked by the recombinant zebrafish Igf3 protein. Intriguingly, the phosphorylation of Igf1r, the receptor for Igf3, could be activated by hCG in the follicular cells during ovulation. And inhibition of Igf3 signaling by Igf1r inhibitors and Igf3 antiserum could significantly attenuate the hCG-induced ovulation. Collectively, all these data support the notion that Igf3 serves as a mediator of LH action in zebrafish ovulation.
Assuntos
Hormônio Luteinizante/metabolismo , Ovulação/fisiologia , Somatomedinas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Gonadotropina Coriônica/administração & dosagem , Gonadotropina Coriônica/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Somatomedinas/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
The pivotal role of androgen receptor (AR) in regulating male fertility has attracted much research attention in the past two decades. Previous studies have shown that total AR knockout would lead to incomplete spermatogenesis and lowered serum testosterone levels in mice, resulting in azoospermia and infertility. However, the precise physiological role of ar in controlling fertility of male fish is still poorly understood. In this study, we have established an ar knockout zebrafish line by transcription activator-like effectors nucleases. Homozygous ar mutant male fish with smaller testis size were found to be infertile when tested by natural mating. Intriguingly, a small amount of mature spermatozoa was observed in the ar mutant fish. These mature spermatozoa could fertilize healthy oocytes, albeit with a lower fertilization rate, by in vitro fertilization. Moreover, the expression levels of most steroidogenic genes in the testes were significantly elevated in the ar mutants. In contrast, the levels of estradiol and 11-ketotestosterone (11-KT) were significantly decreased in the ar mutants, indicating that steroidogenesis was defective in the mutants. Furthermore, the protein level of LHß in the serum decreased markedly in the ar mutants when compared with wild-type fish, probably due to the positive feedback from the diminished steroid hormone levels.
Assuntos
Fertilidade/genética , Infertilidade Masculina/genética , Receptores Androgênicos/genética , Espermatogênese/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Estradiol/metabolismo , Infertilidade Masculina/metabolismo , Hormônio Luteinizante Subunidade beta/metabolismo , Masculino , Receptores Androgênicos/metabolismo , Células de Sertoli/metabolismo , Testículo/metabolismo , Testosterona/análogos & derivados , Testosterona/metabolismo , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: The purpose of this study was to evaluate the association between single-nucleotide polymorphisms and chronic postsurgical pain. METHODS: Using GoldenGate genotyping assays, we genotyped 638 polymorphisms within 54 pain-related genes in 1,152 surgical patients who were enrolled in our Persistent Pain after Surgery Study. Patients were contacted by phone to determine whether they had chronic postsurgical pain at 12 months. Polymorphisms identified were validated in a matched cohort of 103 patients with chronic postsurgical pain and 103 patients who were pain free. The functions of targeted polymorphisms were tested in an experimental plantar incisional nociception model using knock-in mice. RESULTS: At 12 months after surgery, 246 (21.4%) patients reported chronic postsurgical pain. Forty-two polymorphisms were found to be associated with chronic postsurgical pain, 19 decreased the risk of pain, and 23 increased the risk of pain. Patients carrying allele A of rs6265 polymorphism in brain-derived neurotrophic factor (BDNF) had a lower risk of chronic postsurgical pain in the discovery and validation cohorts, with an adjusted odds ratio (95% CI) of 0.62 (0.43 to 0.90) and 0.57 (0.39 to 0.85), respectively. Age less than 65 yr, male sex, and prior history of pain syndrome were associated with an increased risk of pain. Genetic polymorphisms had higher population attributable risk (7.36 to 11.7%) compared with clinical risk factors (2.90 to 5.93%). Importantly, rs6265 is a substitution of valine by methionine at amino acid residue 66 (Val66Met) and was associated with less mechanical allodynia in BDNF mice compared with BDNF group after plantar incision. CONCLUSIONS: This study demonstrated that genetic variant of BDNF rs6265G>A is associated with decreased risk of chronic postsurgical pain.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Dor Crônica/genética , Técnicas de Genotipagem , Dor Pós-Operatória/genética , Polimorfismo de Nucleotídeo Único/genética , Animais , Estudos de Coortes , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Camundongos , Pessoa de Meia-IdadeRESUMO
Zebrafish is an important model to study developmental biology and human diseases. However, an effective approach to achieve spatial and temporal gene knockout in zebrafish has not been well established. In this study, we have developed a new approach, namely bacterial artificial chromosome-rescue-based knockout (BACK), to achieve conditional gene knockout in zebrafish using the Cre/loxP system. We have successfully deleted the DiGeorge syndrome critical region gene 8 (dgcr8) in zebrafish germ line and demonstrated that the maternal-zygotic dgcr8 (MZdgcr8) embryos exhibit MZdicer-like phenotypes with morphological defects which could be rescued by miR-430, indicating that canonical microRNAs play critical role in early development. Our findings establish that Cre/loxP-mediated tissue-specific gene knockout could be achieved using this BACK strategy and that canonical microRNAs play important roles in early embryonic development in zebrafish.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Técnicas de Inativação de Genes/métodos , Células Germinativas/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Sequência de Bases , Desenvolvimento Embrionário/genética , Éxons/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação/genética , Processamento Pós-Transcricional do RNA/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
The aim of this study is to investigate the functional role of G protein-coupled receptor 30 (GPR30) in the epididymis. We found that GPR30 is expressed in the epithelium of the immature rat epididymis and is involved in chloride secretion into the caudal epididymis lumen. The short-circuit current (Isc) experiments showed that in primary cultured caudal epididymis epithelium, activation of GPR30 by its specific agonist G1 induced a mono-phasic current increase, and G15, the specific antagonist of GPR30, could completely inhibit the current induced by G1. The G1-induced Isc was largely blocked by application of the non-specific chloride channel inhibitor diphenylamine-dicarboxylic acid (DPC), or by the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172 , suggesting that the current was mainly mediated through CFTR. In addition, after stimulating GPR30 by G1, the intracellular concentration of cAMP in the epithelium was significantly increased, indicating that the cAMP signal pathway is involved and could be responsible for the CFTR activation. Finally, to further investigate the function of GPR30 in vivo, G15 was administrated into rats subcutaneously. The osmotic pressure of the micro perfusion solution from epididymis was measured and the sperms were collected. Results showed that there was an osmotic pressure increase of the perfusion solution from G15 treated rats. When the GPR30 was inhibited by G15 endogenously, the motility of sperms decreased. Our data demonstrated that GPR30 is involved in the formation of caudal epididymis fluid micro-environment thus affecting sperm motility.
Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Receptores Acoplados a Proteínas G/genética , Animais , Western Blotting , Células Cultivadas , Cloretos/metabolismo , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Epididimo/citologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Microscopia de Fluorescência , Pressão Osmótica/efeitos dos fármacos , Técnicas de Patch-Clamp , Quinolinas/química , Quinolinas/farmacologia , Quinolonas/química , Quinolonas/farmacologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Motilidade dos Espermatozoides/efeitos dos fármacosRESUMO
BACKGROUND: Proteases have been shown to modulate pain signaling in the spinal cord and may contribute to the development of chronic postsurgical pain. By using peripheral inflammation in rats as a chronic pain model, the authors identified the deregulation of proteases and their inhibitors as a hallmark of chronic pain development using a genome-wide screening approach. METHODS: A microarray analysis was performed and identified spinal cathepsin G (CTSG) as the most up-regulated gene in rats with persistent hyperalgesia after intraplantar injection of complete Freund's adjuvant (n = 4). Further experiments were performed to elucidate the mechanisms of CTSG-induced hyperalgesia by intrathecally applying specific CTSG inhibitor (n = 10). The authors also evaluated the association between CTSG gene polymorphisms and the risk of chronic postsurgical pain in 1,152 surgical patients. RESULTS: CTSG blockade reduced heat hyperalgesia, accompanied by a reduction in neutrophil infiltration and interleukin 1ß levels in the dorsal horns. In the gene association study, 246 patients (21.4%) reported chronic postsurgical pain at 12-month follow-up. Patients with AA genotypes at polymorphisms rs2070697 (AA-15.3%, GA-24.1%, and GG-22.3%) or rs2236742 (AA-6.4%, GA-20.4%, and GG-22.6%) in the CTSG gene had lower risk for chronic postsurgical pain compared with wild-types. The adjusted odds ratios were 0.67 (95% CI, 0.26 to 0.99) and 0.34 (95% CI, 0.21 to 0.98), respectively. CONCLUSIONS: This study demonstrated that CTSG is a pronociceptive mediator in both animal model and human study. CTSG represents a new target for pain control and a potential marker to predict patients who are prone to develop chronic pain after surgery.
Assuntos
Catepsina G/biossíntese , Dor Crônica/metabolismo , Dor Pós-Operatória/metabolismo , Idoso , Animais , Catepsina G/genética , Células Cultivadas , Dor Crônica/genética , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição da Dor/métodos , Dor Pós-Operatória/genética , Ratos , Ratos Sprague-Dawley , Regulação para Cima/fisiologiaRESUMO
Transcription activator like effector nucleases (TALENs) is a promising approach to disrupt intended genomic loci. The assembly of highly effective TALENs is critical for successful genome editing. Recently we reported a convenient and robust platform to construct customized TALENs. The TALENs generated by this platform have been proven to be highly effective for gene disruption in Xenopus tropicalis and zebrafish as well as large genomic deletions in zebrafish. The one-time success rate of targeted gene disruption is about 90% for more than 100 genomic loci tested, with the mutation frequencies often reaching above 50%. Here we describe the validated protocol for TALEN assembly, methods for generating gene knockout animals in X. tropicalis and zebrafish, as well as the protocol for engineering large genomic deletions in zebrafish.
Assuntos
Técnicas de Inativação de Genes , Xenopus/genética , Peixe-Zebra/genética , Animais , Desoxirribonucleases/genética , Deleção de Genes , Engenharia Genética/métodos , Genoma , Microinjeções , Xenopus/embriologia , Peixe-Zebra/embriologiaRESUMO
Transcription activator-like effector nucleases (TALENs) are an approach for directed gene disruption and have been proved to be effective in various animal models. Here, we report that TALENs can induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germ-line transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for RNA transcription and microinjection into Xenopus embryos. Eight pairs of TALENs were constructed to target eight Xenopus genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci. Furthermore, mutations induced by TALENs were highly efficiently passed through the germ line to F(1) frogs. Together with simple and reliable PCR-based approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus.
Assuntos
Endonucleases/metabolismo , Marcação de Genes , Transativadores/metabolismo , Xenopus/embriologia , Animais , Sequência de Bases , DNA , Embrião não Mamífero , Dados de Sequência Molecular , Xenopus/genéticaRESUMO
This paper describes comparative studies and protocols in (1) self-assembling of ultrasmall superparamagnetic iron oxide nanoparticle (NP), circular plasmid DNA, and branched polyethylenimine (PEI) composites; (2) magnetofection; (3) gene delivery, (4) magnetic resonance imaging (MRI), and (5) cytotoxicity of the composites toward hepatocellular carcinoma HepG2 cells.
Assuntos
Nanocompostos/química , Coloração e Rotulagem , Alginatos/química , Animais , Carcinoma Hepatocelular , Sobrevivência Celular , Citratos/química , DNA Circular/química , DNA Circular/genética , Cães , Óxido Ferroso-Férrico/química , Proteínas de Fluorescência Verde/biossíntese , Células Hep G2 , Humanos , Luciferases de Renilla/biossíntese , Células Madin Darby de Rim Canino , Imageamento por Ressonância Magnética , Plasmídeos/química , Plasmídeos/genética , Polietilenoimina/química , TransfecçãoRESUMO
Searching for depigmenting agents from natural sources has become a new direction in the cosmetic industry as natural products are generally perceived as relatively safer. In our previous study, selected Chinese medicines traditionally used to treat hyperpigmentation were tested for anti-hyperpigmentary effects using a melan-a cell culture model. Among the tested chemical compounds, 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were found to possess hypopigmentary effects. Western blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), cyclic adenosine monophosphate (cAMP) assay, protein kinase A (PKA) activity assay, tyrosinase inhibition assay and lipid peroxidation inhibition assay were performed to reveal the underlying cellular and molecular mechanisms of the hypopigmentary effects. 4-Ethylresorcinol and 4-ethylphenol attenuated mRNA and protein expression of tyrosinase-related protein (TRP)-2, and possessed antioxidative effect by inhibiting lipid peroxidation. 1-Tetradecanol was able to attenuate protein expression of tyrosinase. The hypopigmentary actions of 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were associated with regulating downstream proteins along the PKA pathway. 4-Ethylresorcinol was more effective in inhibiting melanin synthesis when compared to 4-ethylphenol and 1-tetradecanol.
Assuntos
Hiperpigmentação/metabolismo , Animais , Western Blotting , Linhagem Celular , AMP Cíclico/metabolismo , Hiperpigmentação/genética , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Fenóis/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A hybrid composite made up of superparamagnetic iron oxide nanoshells encapsulating the anticancer drug doxorubicin and bound together by poly(vinyl alcohol) was developed. Transcatheter arterial delivery in an inâ vivo liver tumor model led to embolization of the liver tumor blood vessels. Embolization was followed by disassembly of the composite. The nanoshells were then able to pass through the leaky tumor vasculature into the tumor tissue, thereby leading to slow and sustained release of the drug. As well as being relatively noncytotoxic, the composite was responsive to magnetic resonance imaging, thus making it a potentially useful theranostic agent.
Assuntos
Quimioembolização Terapêutica , Doxorrubicina/administração & dosagem , Compostos Férricos/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Imageamento por Ressonância Magnética/métodos , Nanoestruturas , Álcool de Polivinil/administração & dosagem , Animais , CoelhosRESUMO
Gonadotropin-releasing hormone (GnRH) is a key stimulator for gonadotropin secretion in the pituitary and its pivotal role in reproduction is well conserved in vertebrates. In fish models, GnRH can also induce prolactin (PRL) release, but little is known for the corresponding effect on PRL gene expression as well as the post-receptor signalling involved. Using grass carp as a model, the functional role of GnRH and its underlying signal transduction for PRL regulation were examined at the pituitary level. Using laser capture microdissection coupled with RT-PCR, GnRH receptor expression could be located in carp lactotrophs. In primary cell culture prepared from grass carp pituitaries, the native forms of GnRH, GnRH2 and GnRH3, as well as the GnRH agonist [D-Arg6, Pro9, NEt]-sGnRH were all effective in elevating PRL secretion, PRL mRNA level, PRL cell content and total production. In pituitary cells prepared from the rostral pars distalis, the region in the carp pituitary enriched with lactotrophs, GnRH not only increased cAMP synthesis with parallel CREB phosphorylation and nuclear translocation but also induced a rapid rise in cytosolic Ca2+ by Ca2+ influx via L-type voltage-sensitive Ca2+ channel (VSCC) with subsequent CaM expression and NFAT2 dephosphorylation. In carp pituitary cells prepared from whole pituitaries, GnRH-induced PRL secretion was reduced/negated by inhibiting cAMP/PKA, PLC/PKC and Ca2+/CaM/CaMK-II pathways but not the signalling events via IP3 and CaN/NFAT. The corresponding effect on PRL mRNA expression, however, was blocked by inhibiting cAMP/PKA/CREB/CBP and Ca2+/CaM/CaN/NFAT2 signalling but not PLC/IP3/PKC pathway. At the pituitary cell level, activation of cAMP/PKA pathway could also induce CaM expression and Ca2+ influx via VSCC with parallel rises in PRL release and gene expression in a Ca2+/CaM-dependent manner. These findings, as a whole, suggest that the cAMP/PKA-, PLC/PKC- and Ca2+/CaM-dependent cascades are differentially involved in GnRH-induced PRL secretion and PRL transcript expression in carp lactotrophs. During the process, a functional crosstalk between the cAMP/PKA- and Ca2+/CaM-dependent pathways may occur with PRL release linked with CaMK-II and PKC activation and PRL gene transcription caused by nuclear action of CREB/CBP and CaN/NFAT2 signalling.
Assuntos
Cálcio , Carpas , Proteínas Quinases Dependentes de AMP Cíclico , AMP Cíclico , Hormônio Liberador de Gonadotropina , Hipófise , Prolactina , Proteína Quinase C , Fosfolipases Tipo C , Animais , Carpas/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Prolactina/metabolismo , Hipófise/metabolismo , Hipófise/citologia , Proteína Quinase C/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cálcio/metabolismo , Fosfolipases Tipo C/metabolismo , Fosfolipases Tipo C/genética , AMP Cíclico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Calmodulina/metabolismo , Células Cultivadas , Expressão Gênica/efeitos dos fármacosRESUMO
Cardiac glycosides as inhibitors of the sodium/potassium adenosine triphosphatase (sodium pump) have been reported to block cancer growth by inducing G2/M phase arrest in many cancer cells. However, no detailed studies have been performed to distinguish between these two phases of cardiac glycoside-arrested cells. Furthermore, the underlying mechanisms involved in this cell cycle arrest process are still not known. Here, we report that bufalin and other cardiac glycosides potently induce mitotic arrest by the downregulation of polo-like kinase 1 (Plk1) expression. Live-cell imaging results demonstrate that bufalin-treated cells exhibit a marked delay in entering prophase at an early stage and are then arrested at prometaphase or induced entry into apoptosis. This phenotypic change is attributed to the downregulation of Plk1. We also show that bufalin and the knockdown of sodium pump reduce Plk1, at least in part, through downregulation of the nuclear transcription factors, hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB). These findings suggest that cardiac glycosides induce mitotic arrest and apoptosis through HIF-1α- and NF-κB-mediated downregulation of Plk1 expression, demonstrating that HIF-1α and NF-κB are critical targets of cardiac glycosides in exerting their anticancer action.