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OBJECTIVE: Efficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine ß-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). METHODS: Bovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine ß-casein gene locus, followed by treatment of the cells with CdCl2 for 24 hours. After 3 days of CdCl2 treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR). RESULTS: Treatment with CdCl2 decreased the mRNA expression of RAD51 and MMR-related genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency. CONCLUSION: Treatment with CdCl2 inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.
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SCOPE: Obesity and diabetes are major public health problems and are emerging as pandemics. Considerable evidence suggests that pear fruit consumption is associated with a lower risk of obesity-related complications. Thus, the present study is conducted to investigate the therapeutic potential of pear extract (PE) for reversing obesity and associated metabolic complications in high-fat diet-induced obese mice. METHODS AND RESULTS: Obesity is induced in male C57BL/6 mice fed a high-fat diet for 11 weeks. After the first 6 weeks on the diet, obese mice are administered vehicle or PE for 5 weeks. PE treatment decreases body weight gain, expands white adipose tissue (WAT), and causes hepatic steatosis in obese mice, as well as inhibits adipogenesis and lipogenesis. Impaired glucose tolerance and insulin resistance are improved by PE. In addition, PE reduces macrophage infiltration and expression of pro-inflammatory genes and deactivates mitogen-activated protein kinases in WAT. Finally, malaxinic acid is identified as an active component responsible for the anti-obesity effects of PE in mice. CONCLUSION: The results demonstrate that PE supplementation ameliorates diet-induced obesity and associated metabolic complications and suggest the health-beneficial effects of both pear fruits and malaxinic acid in counteracting these diseases.
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Fármacos Antiobesidade/uso terapêutico , Benzoatos/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Obesidade/dietoterapia , Paniculite/dietoterapia , Extratos Vegetais/farmacologia , Piranos/uso terapêutico , Pyrus/química , Adipogenia/efeitos dos fármacos , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/patologia , Animais , Fármacos Antiobesidade/farmacologia , Benzoatos/farmacologia , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Obesidade/etiologia , Paniculite/etiologia , Paniculite/patologia , Extratos Vegetais/análise , Polifenóis/análise , Piranos/farmacologia , Aumento de Peso/efeitos dos fármacosRESUMO
Trimethyltin (TMT), a potent neurotoxic chemical, causes dysfunction and neuroinflammation in the brain, particularly in the hippocampus. The present study assessed TMT-induced glial cell activation and inflammatory cytokine alterations in the mouse hippocampus, BV-2 microglia, and primary cultured astrocytes. In the mouse hippocampus, TMT treatment significantly increased the expression of glial cell markers, including the microglial marker ionized calcium-binding adapter molecule 1 and the astroglial marker glial fibrillary acidic protein. The expression of M1 and M2 microglial markers (inducible nitric oxide synthase [iNOS] and CD206, respectively) and pro-inflammatory cytokines (interleukin [IL]-1ß, IL-6 and tumor necrosis factor [TNF]-α) were significantly increased in the mouse hippocampus following TMT treatment. In BV-2 microglia, iNOS, IL-1ß, TNF-α, and IL-6 expression increased significantly, whereas arginase-1 and CD206 expression decreased significantly after TMT treatment in a time- and concentration-dependent manner. In primary cultured astrocytes, iNOS, arginase-1, IL-1ß, TNF-α, and IL-6 expression increased significantly, whereas IL-10 expression decreased significantly after TMT treatment in a time- and concentration-dependent manner. These results indicate that significant up-regulation of pro-inflammatory signals in TMT-induced neurotoxicity may be associated with pathological processing of TMT-induced neurodegeneration.
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Astrócitos/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Microglia/efeitos dos fármacos , Compostos de Trimetilestanho/farmacologia , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/metabolismoRESUMO
Individuals with cancer are particularly susceptible to depression and cognitive impairment. However, the precise mechanisms underlying cancer-induced hippocampal dysfunction are poorly understood. We investigated the effects of a peripheral tumor on emotional behavior, hippocampus-dependent memory and associated molecular and cellular features using an experimental animal model. Behavioral alterations were examined; stress-related parameters measured; hippocampal neurogenesis evaluated; and the levels of pro-inflammatory cytokines, brain-derived neurotrophic factor (BDNF) and cyclooxygenase-2 (COX-2) assayed, 2 weeks after inoculation of adult BALB/c mice with cells of a colon carcinoma cell line (CT26). As the tumors developed, CT26-inoculated mice showed significant increases in the depression-like behavior (measured using the tail suspension test) and memory impairment (in terms of object recognition) compared with vehicle-inoculated controls. The presence of a peripheral tumor significantly elevated the hippocampal levels of mRNAs encoding interleukin-6 (IL-6) and tumor necrosis factor-α, as well as plasma IL-6 and corticosterone levels. Additionally, the adrenal glands became enlarged, and the numbers of Ki-67-positive proliferating hippocampal cells and doublecortin-positive immature progenitor neurons, as well as the constitutive levels of mRNAs encoding BDNF and COX-2 were significantly reduced. Therefore, a peripheral tumor alone may be sufficient to induce hippocampal dysfunction, possibly by reducing the rate of neurogenesis and the levels of BDNF and COX-2 in that tissue and also by increasing stress-related parameters and the circulating levels of pro-inflammatory cytokines.
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Hipocampo/metabolismo , Neoplasias Experimentais/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Linhagem Celular Tumoral , Corticosterona/sangue , Ciclo-Oxigenase 2/metabolismo , Citocinas/sangue , Citocinas/metabolismo , Depressão/metabolismo , Feminino , Hipocampo/patologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neurogênese/fisiologia , Reconhecimento Psicológico/fisiologia , Estresse FisiológicoRESUMO
The generation of induced pluripotent stem (iPS) cells is a powerful tool in regenerative medicine, and advances in nanotechnology clearly have great potential to enhance stem cell research. Here, we introduce a liposomal magnetofection (LMF) method for iPS cell generation. Efficient conditions for generating virus-free iPS cells from mouse embryonic fibroblast (MEF) cells were determined through the use of different concentrations of CombiMag nanoparticle-DNA(pCX-OKS-2A and pCX-cMyc)-lipoplexes and either one or two cycles of the LMF procedure. The cells were prepared in a short reprogramming time period (≤ 8 days, 0.032-0.040%). Among the seven LMF-iPS cell lines examined, two were confirmed to be integration-free, and an integration-free LMF-iPS cell line was produced under the least toxic conditions (single LMF cycle with a half-dose of plasmid). This cell line also displayed in vitro/in vivo pluripotency, including teratoma formation and chimeric mouse production. In addition, the safety of CombiMag-DNA lipoplexes for the transfection of MEF cells was confirmed through lactate dehydrogenase activity assay and transmission electron microscopy. These results demonstrated that the LMF method is simple, effective, and safe. LMF may represent a superior technique for the generation of virus-free or integration-free iPS cell lines that could lead to enhanced stem cell therapy in the future.
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Técnicas de Cultura de Células , Células-Tronco Pluripotentes Induzidas/citologia , Lipossomos/metabolismo , Animais , Células Cultivadas , DNA/química , DNA/metabolismo , Técnicas Genéticas , L-Lactato Desidrogenase/metabolismo , Magnetismo , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Modelos Genéticos , Plasmídeos/metabolismo , Células-Tronco/citologia , Teratoma/metabolismo , Fatores de Tempo , TransgenesRESUMO
We demonstrate the regulation of OCT4 gene expression mediated by liver receptor homolog-1 (LRH-1) in human embryonic carcinoma cells. LRH-1 and OCT4 are co-expressed in undifferentiated NCCIT cells and decreased during retinoic acid-induced differentiation. Dose-dependent overexpression of LRH-1 transactivated the OCT4 promoter activity and its dominant negative form with a deletion of activation function-2 motif reduced the activity even in the presence of LRH-1. The OCT4 promoter contains potent three LRH-1 binding sites; one within conserved region (CR) 1 and two within CR2. Mutagenesis of each binding site affected the decrease in OCT4 promoter activity and the 2nd binding site mutant most significantly reduced the transcriptional activity, compared to that of 1st and 3rd binding site mutants, respectively. Simultaneous disruption of 2nd and 3rd binding sites led to significant down-regulation of the activity even in the presence of 1st binding site-containing CR1. Moreover, mutation of each binding element within native or exogenous minimal promoter-driven CR1 or CR2 also decreased the promoter activity to some different extent, suggesting that three binding elements may be implicated in the induction of the full-activity of OCT4 promoter. In vivo binding assay revealed the 2nd and 3rd binding motifs within CR2 were more enriched than the 1st one within CR1. Taken together, our study indicates that LRH-1 acts as a transcriptional activator in the regulation of OCT4 gene expression through the cooperative interaction with three binding sites directly or/and indirectly.
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Carcinoma Embrionário/genética , Regulação Neoplásica da Expressão Gênica , Fator 3 de Transcrição de Octâmero/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Ativação Transcricional , Sítios de Ligação , Carcinoma Embrionário/patologia , Linhagem Celular Tumoral , Humanos , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/genética , Transcrição Gênica , Tretinoína/farmacologiaRESUMO
The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Milk is presently the most mature system for production of therapeutic proteins from a transgenic animal. Specifically, ß-casein is a major component of cow, goat and sheep milk, and its promoter has been used to regulate the expression of transgenic genes in the mammary gland of transgenic animals. Here, we cloned the porcine ß-casein gene and analyzed the transcriptional activity of the promoter and intron 1 region of the porcine ß-casein gene. Sequence inspection of the 5'-flanking region revealed potential DNA elements including SRY, CdxA, AML-a, GATA-3, GATA-1 and C/EBP ß. In addition, the first intron of the porcine ß-casein gene contained the transcriptional enhancers Oct-1, SRY, YY1, C/EBP ß, and AP-1, as well as the retroviral TATA box. We estimated the transcriptional activity for the 5'-proximal region with or without intron 1 of the porcine ß-casein gene in HC11 cells stimulated with lactogenic hormones. High transcriptional activity was obtained for the 5'-proximal region with intron 1 of the porcine ß-casein gene. The ß-casein gene containing the mutant TATA box (CATAAAA) was also cloned from another individual pig. Promoter activity of the luciferase vector containing the mutant TATA box was weaker than the same vector containing the normal TATA box. Taken together, these findings suggest that the transcription of porcine ß-casein gene is regulated by lactogenic hormone via intron 1 and promoter containing a mutant TATA box (CATAAAA) has poor porcine ß-casein gene activity.
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Human papillomavirus E6 forms a complex with p53 tumor suppressor and E6-associated protein, leading to the degradation of p53 via the ubiquitination pathway, resulting in the oncogenesis of cervical carcinomas. Several viral and cellular gene promoters were shown to be transactivated by E6 oncogene. In this study, to understand the role of transcription activity of E6 related to cervical carcinogenesis, the effect of cervical cancer drugs on E6 induced transcription activity has been elucidated. Several viral promoter (RSV, CMV, SV40, and HIV)-luciferase reporter gene constructs, and eukaryotic E6 expression vector were prepared as an E6 transcription analysis system and an exogenous E6 protein source, respectively. It was shown that the promoters of RSV, SV40, and HIV, but not CMV, were transactivated by HPV 16 E6 in cervical cancer cell line. Several known cervical cancer drugs were investigated for their effects on transcription activity of E6 in SiHa stably transfected with E6-responsive promoters. Cervical cancer drugs consistently reduced luciferase activity, in transfectants with RSV-luc (SiHa/pRSV-luc, KCTC 0427BP) E6 mRNA also. Thus, in this study, we have demonstrated that the promoters of RSV, HIV, and SV40 were transactivated by E6 in cervical cancer cells. Three cervical cancer drugs downregulated RSV-luc transcription and E6 expression by a p53 independent pathway. RSV-luc promoter analysis system could be useful for understanding the role of transcription activity of E6 related to cervical cancer and also for screening drugs against cervical cancers caused by HPV infection.