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1.
BMC Genomics ; 21(1): 625, 2020 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-32942992

RESUMO

BACKGROUND: Parkinson's disease (PD) is a degenerative disease with early-stage pathology hypothesized to manifest in brainstem regions. Vocal deficits, including soft, monotone speech, result in significant clinical and quality of life issues and are present in 90% of PD patients; yet the underlying pathology mediating these significant voice deficits is unknown. The Pink1-/- rat is a valid model of early-onset PD that presents with analogous vocal communication deficits. Previous work shows abnormal α-synuclein protein aggregation in the periaqueductal gray (PAG), a brain region critical and necessary to the modulation of mammalian vocal behavior. In this study, we used high-throughput RNA sequencing to examine gene expression within the PAG of both male and female Pink1-/- rats as compared to age-matched wildtype controls. We used a bioinformatic approach to (1) test the hypothesis that loss of Pink1 in the PAG will influence the differential expression of genes that interact with Pink1, (2) highlight other key genes that relate to this type of Mendelian PD, and (3) catalog molecular targets that may be important for the production of rat vocalizations. RESULTS: Knockout of the Pink1 gene resulted in differentially expressed genes for both male and female rats that also mapped to human PD datasets. Pathway analysis highlighted several significant metabolic pathways. Weighted gene co-expression network analysis (WGCNA) was used to identify gene nodes and their interactions in (A) males, (B) females, and (C) combined-sexes datasets. For each analysis, within the module containing the Pink1 gene, Pink1 itself was the central node with the highest number of interactions with other genes including solute carriers, glutamate metabotropic receptors, and genes associated with protein localization. Strong connections between Pink1 and Krt2 and Hfe were found in both males and female datasets. In females a number of modules were significantly correlated with vocalization traits. CONCLUSIONS: Overall, this work supports the premise that gene expression changes in the PAG may contribute to the vocal deficits observed in this PD rat model. Additionally, this dataset identifies genes that represent new therapeutic targets for PD voice disorders.


Assuntos
Redes Reguladoras de Genes , Doença de Parkinson/genética , Substância Cinzenta Periaquedutal/metabolismo , Transcriptoma , Vocalização Animal , Animais , Feminino , Proteína da Hemocromatose/genética , Proteína da Hemocromatose/metabolismo , Queratina-2/genética , Queratina-2/metabolismo , Masculino , Doença de Parkinson/metabolismo , Substância Cinzenta Periaquedutal/fisiologia , Proteínas Quinases/genética , Ratos , Ratos Long-Evans
2.
Sci Rep ; 10(1): 4829, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32179842

RESUMO

Abnormal keratinocyte differentiation is fundamental to pathologies such as skin cancer and mucosal inflammatory diseases. The ability to grow keratinocytes in vitro allows the study of differentiation however any translational value is limited if keratinocytes get altered by the culture method. Although serum lipids (SLPs) and phenol red (PR) are ubiquitous components of culture media their effect on differentiation is largely unknown. We show for the first time that PR and SLP themselves suppress expression of differentiation-specific keratins K1, K10 and K2 in normal human epidermal keratinocytes (NHEK) and two important cell lines, HaCaT and N/TERT-1. Removal of SLP increased expression of K1, K10 and K2 in 2D and 3D cultures, which was further enhanced in the absence of PR. The effect was reversed for K1 and K10 by adding all-trans retinoic acid (ATRA) but increased for K2 in the absence of PR. Furthermore, retinoid regulation of differentiation-specific keratins involves post-transcriptional mechanisms as we show KRT2 mRNA is stabilised whilst KRT1 and KRT10 mRNAs are destabilised in the presence of ATRA. Taken together, our results indicate that the presence of PR and SLP in cell culture media may significantly impact in vitro studies of keratinocyte differentiation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Queratina-10/genética , Queratina-10/metabolismo , Queratina-1/genética , Queratina-1/metabolismo , Queratina-2/genética , Queratina-2/metabolismo , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Lipídeos/fisiologia , Fenolsulfonaftaleína/farmacologia , Tretinoína/farmacologia , Células Cultivadas , Células HaCaT , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
3.
Dev Neurobiol ; 77(11): 1308-1320, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28719101

RESUMO

The olfactory epithelium (OE) has the remarkable capability to constantly replace olfactory receptor neurons (ORNs) due to the presence of neural stem cells (NSCs). For this reason, the OE provides an excellent model to study neurogenesis and neuronal differentiation. In the present work, we induced neuronal degeneration in the OE of Xenopus laevis larvae by bilateral axotomy of the olfactory nerves. We found that axotomy induces specific- neuronal death through apoptosis between 24 and 48h post-injury. In concordance, there was a progressive decrease of the mature-ORN marker OMP until it was completely absent 72h post-injury. On the other hand, neurogenesis was evident 48h post-injury by an increase in the number of proliferating basal cells as well as NCAM-180- GAP-43+ immature neurons. Mature ORNs were replenished 21 days post-injury and the olfactory function was partially recovered, indicating that new ORNs were integrated into the olfactory bulb glomeruli. Throughout the regenerative process no changes in the expression pattern of the neurotrophin Brain Derivate Neurotrophic Factor were observed. Taken together, this work provides a sequential analysis of the neurodegenerative and subsequent regenerative processes that take place in the OE following axotomy. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1308-1320, 2017.


Assuntos
Axotomia , Degeneração Neural/etiologia , Degeneração Neural/patologia , Mucosa Olfatória/patologia , Traumatismos do Nervo Olfatório/patologia , Regeneração/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Caspase 3/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Proteína GAP-43/metabolismo , Regulação da Expressão Gênica/fisiologia , Queratina-2/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Proteína de Marcador Olfatório/metabolismo , Traumatismos do Nervo Olfatório/etiologia , Recuperação de Função Fisiológica/fisiologia , Olfato/fisiologia , Fatores de Tempo , Xenopus laevis
4.
Acta Histochem ; 118(5): 505-12, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27265811

RESUMO

In order to investigate the effects of the keratin 2 (KRT2) on alpaca melanocyte in vivo and vitro, the immunohistochemistry (IHC), quantitative real-time PCR (qPCR), Western blot, and alpaca melanocytes transfection methods were used. The results showed that mRNA and protein expression of KRT2 was highly expressed in brown skin in comparison with that in white skin. Moreover, we found that KRT2 was expressed in alpaca melanocytes in vitro by immunocytochemistry. After transfection with KRT2 in alpaca melanocytes, the relative mRNA and protein expression of KRT2, microphthalmia-associtated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1) in alpaca skin melanocytes was increased with significant differences; a further result was the increase of melanin production. The results suggested that KRT2 functions in alpaca hair color formation, which offered an essential theoretical basis for further exploration of the role of melanogenesis.


Assuntos
Queratina-2/metabolismo , Melaninas/biossíntese , Pele/metabolismo , Animais , Camelídeos Americanos , Expressão Gênica , Cor de Cabelo , Queratina-2/genética , Melanócitos/metabolismo , Pele/citologia , Pigmentação da Pele
5.
Free Radic Biol Med ; 73: 75-83, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24838180

RESUMO

The nitration of proteins results from the vigorous production of reactive nitrogen species in inflammatory disease. We previously reported the proteomic analysis of nitrated tryptophan residues in in vitro model cells for inflammatory diseases using a 6-nitrotryptophan-specific antibody. In this paper, we applied this method to the analysis of a disease model animal and identified the 6-nitrotryptophan-containing proteins in the skin of atopic dermatitis model mice (AD-NC/Nga mice). We found three nitrotryptophan-containing proteins, namely, carbonic anhydrase III (CAIII), α-enolase (α-ENO), and cytoskeletal keratin type II (KTII), and identified the positions of the nitrotryptophan residues in their amino acid sequences: Trp47 and Trp123 in CAIII, Trp365 in α-ENO, and Trp221 in KTII. Among these, the nitration of CAIII was increased not only in the lesional skin of AD-NC/Nga mice but also in the mice that did not present any symptoms. The in vitro nitration of purified CAIII by peroxynitrite reduced its CO2 hydratase activity in a dose-dependent manner. In addition, we found that CAIII was induced during the differentiation of normal human epidermal keratinocytes. Furthermore, we found the presence of CAIII and the formation of 6-nitrotryptophan-containing proteins in both the lesional and the nonlesional sections of the skin of patients with atopic dermatitis through immunohistochemical staining. This study provides the first demonstration of the formation of 6-nitrotryptophan in human tissues and disease.


Assuntos
Anidrase Carbônica III/metabolismo , Dermatite Atópica/patologia , Queratina-2/metabolismo , Fosfopiruvato Hidratase/metabolismo , Triptofano/análogos & derivados , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Inflamação/imunologia , Inflamação/patologia , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Ácido Peroxinitroso/química , Ratos , Espécies Reativas de Nitrogênio/química , Pele/patologia , Triptofano/química , Triptofano/imunologia , Triptofano/metabolismo
6.
J Invest Dermatol ; 134(10): 2579-2588, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24751727

RESUMO

Keratin K2 is one of the most abundant structural proteins of the epidermis; however, its biological significance has remained elusive. Here we show that suprabasal type II keratins, K1 and K2, are expressed in a mutually exclusive manner at different body sites of the mouse, with K2 being confined to the ear, sole, and tail skin. Deletion of K2 caused acanthosis and hyperkeratosis of the ear and the tail epidermis, corneocyte fragility, increased transepidermal water loss, and local inflammation in the ear skin. The loss of K2 was partially compensated by upregulation of K1 expression. However, a significant portion of K2-deficient suprabasal keratinocytes lacked a regular cytoskeleton and developed massive aggregates of the type I keratin, K10. Aggregate formation, but not hyperkeratosis, was suppressed by the deletion of both K2 and K10, whereas deletion of K10 alone caused clumping of K2 in ear skin. Taken together, this study demonstrates that K2 is a necessary and sufficient binding partner of K10 at distinct body sites of the mouse and that unbalanced expression of these keratins results in aggregate formation.


Assuntos
Dermatite/metabolismo , Hiperceratose Epidermolítica/metabolismo , Queratina-10/metabolismo , Queratina-2/deficiência , Queratina-2/metabolismo , Dermatopatias/metabolismo , Animais , Dermatite/genética , Dermatite/patologia , Modelos Animais de Doenças , Orelha , , Hiperceratose Epidermolítica/genética , Hiperceratose Epidermolítica/patologia , Queratina-1/metabolismo , Queratina-10/genética , Queratina-2/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pele/metabolismo , Pele/patologia , Dermatopatias/genética , Dermatopatias/patologia , Cauda
7.
J Neurosci Res ; 92(2): 206-17, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24254769

RESUMO

ß-Amyloid (Aß) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2-coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac-tau) and tau phosphorylation (P-tau) by inhibiting activation of P300 and GSK3ß. Aß was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aß accumulation was accompanied by increased Ac-tau and P-tau levels. Concomitantly, these cells showed increased P300 and GSK3ß P-Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3-30 µM) and resveratrol (20 µM). Moreover, decreased expression of SIRT1 and its activity by Aß were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 µM, PKA inhibitor), TBCA (20 µM, inhibitor of CK2), and sirtinol (20 µM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP-dependent protein kinase-linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau-related neurodegeneration in the AD brain.


Assuntos
Queratina-2/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Sirtuína 1/biossíntese , Tauopatias/metabolismo , Tetrazóis/farmacologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Western Blotting , Linhagem Celular , Cilostazol , Imunofluorescência , Humanos , Camundongos , Neurônios/metabolismo , Transfecção , Proteínas tau/metabolismo
8.
Exp Dermatol ; 19(7): 674-81, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20456496

RESUMO

Disorders of keratinization are often treated with vitamin A derivatives (retinoids) which affect keratinocyte differentiation, including keratin (KRT) gene expression. In vivo, suprabasal keratinocytes normally express only keratin (K) 1, K2 and K10, but after topical application of all-trans retinoic acid (ATRA), the granular cells will additionally express K4 and K13, i.e. keratins normally present in oral mucosa and in cultured epidermal keratinocytes. To learn more about the retinoid regulation of keratin expression under in vivo-like conditions, we cultured keratinocytes on de-epidermized dermis in only 0.5% serum. These cells produce a normal-looking epidermis that expresses high mRNA levels of KRT1, KRT2 and KRT10, but minimal amounts of KRT4 and KRT13. Addition of ATRA to the medium for 48 h caused a dose-dependent increase in KRT4/KRT13 and a down-regulation of KRT2 mRNA. An increase in K4 protein was also found. The response was greater than the up-regulation of another retinoid-regulated gene, CRABPII. By studying 10 retinoids with different affinities for the retinoic acid receptors (RAR) and retinoid X receptors (RXR) isoforms, the reciprocal expression of KRT2 and KRT4/KRT13 could be connected with agonists for RARalpha. Two of these agonists, CD336/Am580 and CD2081, altered the expression profile with similar potency as the pan-RAR agonists ATRA and CD367. Co-addition of a pan-RAR antagonist (CD3106/AGN193109) markedly inhibited the induction of KRT4/KRT13 expression, whereas the down-regulation of KRT2 was less affected. In conclusion, RARalpha agonists elicit a reciprocal modulation of KRT2 and KRT4/KRT13 expression in human epidermis, but whether or not the keratin genes also possess RARalpha-specific regulatory elements is still unclear.


Assuntos
Queratinas/metabolismo , Receptores do Ácido Retinoico/metabolismo , Retinoides/metabolismo , Retinoides/farmacologia , Pele/efeitos dos fármacos , Pele/metabolismo , Benzoatos/metabolismo , Benzoatos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Queratina-13/genética , Queratina-13/metabolismo , Queratina-2/genética , Queratina-2/metabolismo , Queratina-4/genética , Queratina-4/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/agonistas , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides/metabolismo , Tetra-Hidronaftalenos/metabolismo , Tetra-Hidronaftalenos/farmacologia , Tretinoína/metabolismo , Tretinoína/farmacologia
9.
J Invest Dermatol ; 130(9): 2286-94, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20445547

RESUMO

Loss-of-function mutations in the filaggrin gene are associated with ichthyosis vulgaris and atopic dermatitis. To investigate the impact of filaggrin deficiency on the skin barrier, filaggrin expression was knocked down by small interfering RNA (siRNA) technology in an organotypic skin model in vitro. Three different siRNAs each efficiently suppressed the expression of profilaggrin and the formation of mature filaggrin. Electron microscopy revealed that keratohyalin granules were reduced in number and size and lamellar body formation was disturbed. Expression of keratinocyte differentiation markers and the composition of lipids appeared normal in filaggrin-deficient models. The absence of filaggrin did not render keratins 1, 2, and 10 more susceptible to extraction by urea, arguing against a defect in aggregation. Despite grossly normal stratum corneum morphology, filaggrin-deficient skin models showed a disturbed diffusion barrier function in a dye penetration assay. Moreover, lack of filaggrin led to a reduction in the concentration of urocanic acid, and sensitized the organotypic skin to UVB-induced apoptosis. This study thus demonstrates that knockdown of filaggrin expression in an organotypic skin model reproduces epidermal alterations caused by filaggrin mutations in vivo. In addition, our results challenge the role of filaggrin in intermediate filament aggregation and establish a link between filaggrin and endogenous UVB protection.


Assuntos
Epiderme , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Queratinócitos , Raios Ultravioleta/efeitos adversos , Apoptose/fisiologia , Apoptose/efeitos da radiação , Diferenciação Celular/fisiologia , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Difusão , Células Epidérmicas , Epiderme/metabolismo , Epiderme/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Proteínas Filagrinas , Corantes Fluorescentes/farmacocinética , Humanos , Isoquinolinas/farmacocinética , Queratina-1/metabolismo , Queratina-10/metabolismo , Queratina-2/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Queratinas/metabolismo , Metabolismo dos Lipídeos , Microscopia Eletrônica , Técnicas de Cultura de Órgãos , Permeabilidade , RNA Interferente Pequeno , Solubilidade , Ácido Urocânico/metabolismo
10.
Arch Dermatol Res ; 301(7): 475-85, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19294396

RESUMO

The biosynthesis of retinoic acid (RA) from retinol is controlled by several enzymes, e.g. dehydrogenases (RalDH2, RoDH-4) and retinol-esterifying enzyme (LRAT), whereas its degradation mainly involves CYP26 enzymes. In keratinocytes, RA activates the nuclear retinoid-receptors inducing the transcription of many genes. Here, we examined the effects of RA and the CYP26 inhibitors, liarozole and talarozole, on retinoid metabolism and RA-regulated genes in organotypic epidermis. RA induced the expression of CYP26 enzymes already after 8 h, whereas LRAT exhibited a later response and peaked at 48 h, indicating a feedback induction of retinol esterification. In line with a reduced biosynthesis of RA from retinol after exogenous RA, the expression of RDH16 reduced 80% in response to exogenous RA. The mRNA expression of RA-regulated genes (KRT2, KRT4, CRABPII and HBEGF) was altered within 24 h after RA exposure. In contrast, the CYP26 inhibitors caused only minor effects, except for a clear-cut induction of CYP26A1 only when combined with minute amounts of exogenous RA. Cellular accumulation of exogenous [3H]RA was higher after talarozole than after liarozole, probably indicating a greater CYP26-inhibitory potency of the former drug. The present study shows that CYP26A1 expression is extremely sensitive to both exogenous RA and increased endogenous RA levels, i.e. due to CYP26 inhibition, and thus an excellent biomarker for retinoid signalling in organotypic epidermis.


Assuntos
Aciltransferases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Epiderme/metabolismo , Queratinócitos/metabolismo , Retinal Desidrogenase/metabolismo , Tretinoína/farmacologia , Aciltransferases/genética , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Antagonistas de Androgênios/farmacologia , Benzotiazóis/farmacologia , Biomarcadores/metabolismo , Células Cultivadas , Inibidores das Enzimas do Citocromo P-450 , Epiderme/patologia , Retroalimentação Fisiológica , Regulação Enzimológica da Expressão Gênica , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Imidazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Queratina-2/genética , Queratina-2/metabolismo , Queratina-4/genética , Queratina-4/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Retinal Desidrogenase/genética , Ácido Retinoico 4 Hidroxilase , Tretinoína/metabolismo , Triazóis/farmacologia , Vitamina A/metabolismo
11.
J Am Acad Dermatol ; 57(2): 285-91, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17553594

RESUMO

BACKGROUND: Circumscribed palmar or plantar hypokeratosis (CPH) is a rare skin disorder only recently described. OBJECTIVE: To determine the diagnostic features and to provide insight into the pathogenesis of CPH, with analysis of two new Japanese cases. METHODS: Dermoscopy, immunohistochemistry, electron microscopy, polymerase chain reaction amplification for human papillomavirus (HPV) DNA and 16S microbial rRNA gene profiling were conducted. RESULTS: Dermoscopy showed characteristic features using both dry and jelly immersion observation; step-like desquamation and a homogeneous erythema with regularly distributed whitish spots. Immunohistochemistry revealed strong staining with anti-pankeratin antibody (AE1+AE3) and anti-keratin 16 antibody, and decreased expression of keratin 2e. EM revealed a breakage of the corneocytes within their cytoplasm, but structures for cell attachment were intact. HPV and lesion-specific bacteria were not detected. LIMITATIONS: The number of cases analyzed was two. CONCLUSION: Hyperproliferative epidermal state along with enhanced corneocyte fragility may account for the unique features in CPH.


Assuntos
Dermatoses da Mão/metabolismo , Queratina-2/metabolismo , Queratinas/metabolismo , Ceratose/metabolismo , Idoso , Bactérias/genética , DNA Viral/análise , Feminino , Dermatoses da Mão/microbiologia , Dermatoses da Mão/patologia , Humanos , Imuno-Histoquímica/métodos , Ceratose/microbiologia , Ceratose/patologia , Microscopia Eletrônica , Pessoa de Meia-Idade , Papillomaviridae/genética , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/análise , Coloração e Rotulagem
12.
Differentiation ; 74(4): 186-94, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16683989

RESUMO

We isolated a 2.3 kb DNA segment from the upstream region of the zebrafish cytokeratin II (zfCKII) gene. Transgenic embryos, produced by using a series of 5' deletions linked to the red fluorescent protein (RFP) reporter, showed that the -141/+85 segment of zfCKII directed RFP expression in epidermal cells, whereas the -111/+85 segment did not. When -141/-111 was deleted from -355/+85 and microinjected into one-celled embryos, no fluorescence was observed at later stages, indicating that the -141/-111 segment is required for green fluorescent protein expression in epidermal cells. Furthermore, when a putative KLF-binding site at -119/-117 was mutated, RFP expression rates and intensities were reduced dramatically, although still observed, suggesting that -119/-117 within -141/-111 is a key cis-element for controlling epidermis-specific expression of the zfCKII gene. Finally, we generated a zebrafish transgenic line, Tg(zfCKII(2.3):RFP), which carries an upstream 2.3 kb regulatory region of the zfCKII gene fused with RFP. The expression pattern in the epidermal cells of Tg(zfCKII(2.3):RFP) fish recapitulated that of the endogenous gene. F2 embryos derived from Tg(zfCKII(2.3):RFP) males crossed with wild-type females revealed that the earliest onset of RFP expression was at the sphere stage, indicating that this transgenic approach can be used for studying zygotic expression of maternally inherited genes.


Assuntos
Epiderme/metabolismo , Regulação da Expressão Gênica , Queratina-2/metabolismo , Regiões Promotoras Genéticas , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Células Epidérmicas , Feminino , Genes Reporter , Queratina-2/genética , Masculino , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Distribuição Tecidual , Peixe-Zebra/anatomia & histologia
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