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1.
RNA ; 23(1): 1-5, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27742910

RESUMO

Multiplex genome engineering in vivo with CRISPR/Cas9 shows great promise as a potential therapeutic approach. The ability to incorporate multiple single guide RNA (sgRNA) cassettes together with Cas9 gene expression in one AAV vector could greatly enhance the efficiency. In a recent Method article, Mefferd and coworkers indicated that small tRNA promoters could be used to drive sgRNA expression to facilitate the construction of a more effective AAV vector. In contrast, we found that when targeting endogenous genomic loci, CRISPR/Cas9 with tRNA promoter-driven sgRNA expression showed much reduced genome editing activity, compared with significant cleavage with U6 promoter-driven sgRNA expression. Though the underlying mechanisms are still under investigation, our study suggests that the CRISPR/Cas9 system with tRNA promoter-driven sgRNA expression needs to be reevaluated before it can be used for therapeutic genome editing.


Assuntos
Exorribonucleases/genética , Edição de Genes/métodos , Regiões Promotoras Genéticas , RNA Guia/genética , RNA de Transferência/genética , Sistemas CRISPR-Cas , Expressão Gênica
3.
Yi Chuan ; 37(11): 1167-73, 2015 11.
Artigo em Chinês | MEDLINE | ID: mdl-26582531

RESUMO

The RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has offered a new platform for genome editing with high efficiency. Here, we report the use of CRISPR/Cas9 technology to target a specific genomic region in human pluripotent stem cells. We show that CRISPR/Cas9 can be used to disrupt a gene by introducing frameshift mutations to gene coding region; to knock in specific sequences (e.g. FLAG tag DNA sequence) to targeted genomic locus via homology directed repair; to induce large genomic deletion through dual-guide multiplex. Our results demonstrate the versatile application of CRISPR/Cas9 in stem cell genome editing, which can be widely utilized for functional studies of genes or genome loci in human pluripotent stem cells.


Assuntos
Sistemas CRISPR-Cas/genética , Genoma Humano/genética , Células-Tronco Pluripotentes/metabolismo , Edição de RNA , Mutação da Fase de Leitura , Humanos , RNA Guia/genética
4.
Yi Chuan ; 37(10): 983-91, 2015 10.
Artigo em Inglês | MEDLINE | ID: mdl-26496750

RESUMO

Precision medicine emerges as a new approach that takes into account individual variability. The successful conduct of precision medicine requires the use of precise disease models. Human pluripotent stem cells (hPSCs), as well as adult stem cells, can be differentiated into a variety of human somatic cell types that can be used for research and drug screening. The development of genome editing technology over the past few years, especially the CRISPR/Cas system, has made it feasible to precisely and efficiently edit the genetic background. Therefore, disease modeling by using a combination of human stem cells and genome editing technology has offered a new platform to generate " personalized " disease models, which allow the study of the contribution of individual genetic variabilities to disease progression and the development of precise treatments. In this review, recent advances in the use of genome editing in human stem cells and the generation of stem cell models for rare diseases and cancers are discussed.


Assuntos
Sistemas CRISPR-Cas , Doença/genética , Engenharia Genética/métodos , Genoma Humano/genética , Medicina de Precisão/métodos , Células-Tronco/metabolismo , Humanos , Modelos Genéticos , Neoplasias/genética , Neoplasias/patologia , Medicina de Precisão/tendências , Doenças Raras/genética , Doenças Raras/patologia
5.
Biochem J ; 467(3): 415-24, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25706881

RESUMO

Histone modification plays important roles in many biological processes such as development and carcinogenesis. Methylation of histone H3 lysine 4 (H3K4) is commonly associated with transcriptional activation of genes. H3K4 methylation in mammalian cells is carried out by COMPASS (complex of proteins associated with Set1)-like complexes that are composed of catalytic subunits such as MLL1 (mixed-lineage leukaemia 1) and multiple regulatory subunits in which WDR5 (WD40 repeat-containing protein 5), RBBP5 (retinoblastoma-binding protein 5), ASH2 (absent, small or homoeotic discs 2) and DPY30 [constituting the WRAD sub-complex (WDR5-ASH2-RBBP5-DPY30 complex)] are the major ones shared from yeast to metazoans. We report, in the present paper, a new mode of spatial regulation of H3K4 methyltransferase complexes. PAQR3 (progestin and adipoQ receptors member 3), a tumour suppressor specifically localized in the Golgi apparatus, negatively regulates H3K4 trimethylation (H3K4me3) in mammalian cells. Consistently, HOXC8 and HOXA9 gene expression was negatively regulated by PAQR3 expression levels. Hypoxia-induced H3K4me3 was augmented by PAQR3 knockdown and suppressed by PAQR3 overexpression in AGS gastric cancer cells. PAQR3 was able to interact directly or indirectly with the four members of the WRAD sub-complex and tether them to the Golgi apparatus, accompanied by reduction in histone methyltransferase activity in the nucleus. PAQR3 also interfered with the interaction of WDR5 with the C-terminus of MLL1 (C-ter). Collectively, our study indicates that PAQR3 negatively modulates H3K4 methylation via altering the subcellular compartmentalization of the core regulatory subunits of the COMPASS-like complexes in mammalian cells.


Assuntos
Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Regulação da Expressão Gênica , Genes Homeobox , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Metilação , Modelos Biológicos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
6.
J Nutr ; 144(7): 1009-15, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24828027

RESUMO

Ethanol consumption can lead to hepatic steatosis that contributes to late-stage liver diseases such as cirrhosis and hepatocellular carcinoma. In this study, we investigated the potential protective effect of a flavonoid, luteolin, on ethanol-induced fatty liver development and liver injury. Six-wk-old male C57BL/6 mice were divided into 3 groups: a control group; a group exposed to alcohol by using a chronic and binge ethanol feeding protocol (EtOH); and a group that was administered daily 50 mg/kg of luteolin in addition to ethanol exposure (EtOH + Lut). A chronic and binge ethanol feeding protocol was used, including chronic ethanol consumption (1%, 2%, and 4% for 3 d, and 5% for 9 d) and a binge (30% ethanol) on the last day. Compared with the control group, the EtOH group had a significant elevation in serum concentrations of alanine aminotransferase (ALT) (561%), triglyceride (TG) (47%), and LDL cholesterol (95%), together with lipid accumulation in the liver. Compared with the EtOH group, the EtOH + Lut group had significant reductions in serum concentrations of ALT (43%), TG (22%), LDL cholesterol (52%), and lipid accumulation in the liver. Ethanol elevated liver expression of lipogenic genes including sterol regulatory element-binding protein 1c (Srebp1c) (560%), fatty acid synthase (Fasn) (190%), acetyl-CoA carboxylase (Acc) (48%), and stearoyl-CoA desaturase 1 (Scd1) (286%). Luteolin reduced ethanol-induced expression of these genes in the liver: Srebp1c (79%), Fasn (80%), Acc (60%), and Scd1 (89%). In cultured hepatocytes, luteolin prevented alcohol-induced lipid accumulation and increase in the expression of lipogenic genes. The transcriptional activity of the master regulator of lipid synthesis, sterol regulatory element-binding protein (SREBP), was enhanced by ethanol treatment (160%) and reduced by luteolin administration (67%). In addition, ethanol-induced reduction of AMP-activated protein kinase and SREBP-1c phosphorylation was abrogated by luteolin. Collectively, our study indicates that luteolin is effective in ameliorating ethanol-induced hepatic steatosis and injury.


Assuntos
Alcoolismo/fisiopatologia , Bebedeira/fisiopatologia , Suplementos Nutricionais , Modelos Animais de Doenças , Hepatopatias Alcoólicas/prevenção & controle , Fígado/metabolismo , Luteolina/uso terapêutico , Animais , Antioxidantes/metabolismo , Antioxidantes/uso terapêutico , Linhagem Celular , Etanol/antagonistas & inibidores , Etanol/toxicidade , Fígado Gorduroso Alcoólico/etiologia , Fígado Gorduroso Alcoólico/metabolismo , Fígado Gorduroso Alcoólico/patologia , Fígado Gorduroso Alcoólico/prevenção & controle , Regulação da Expressão Gênica , Hepatite Alcoólica/etiologia , Hepatite Alcoólica/metabolismo , Hepatite Alcoólica/patologia , Hepatite Alcoólica/prevenção & controle , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Lipogênese , Fígado/patologia , Fígado/fisiopatologia , Hepatopatias Alcoólicas/etiologia , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Luteolina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Organismos Livres de Patógenos Específicos
7.
Artigo em Inglês | MEDLINE | ID: mdl-23533476

RESUMO

Wax gourd is a popular vegetable in East Asia. In traditional Chinese medicine, wax gourd peel is used to prevent and treat metabolic diseases such as hyperlipidemia, hyperglycemia, obesity, and cardiovascular disease. However, there is no experimental evidence to support these applications. Here, we examined the effect of the extract of wax gourd peel (EWGP) on metabolic disorders in diet-induced C57BL/6 obese mice. In the preventive experiment, EWGP blocked body weight gain and lowered serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), liver TG and TC contents, and fasting blood glucose in mice fed with a high-fat diet. In the therapeutic study, we induced obesity in the mice and treated with EWGP for two weeks. We found that EWGP treatment reduced serum and liver triglyceride (TG) contents and fasting blood glucose and improved glucose tolerance in the mice. Reporter assay and gene expression analysis showed that EWGP could inhibit peroxisome proliferator-activated receptor γ (PPAR γ ) transactivities and could decrease mRNA levels of PPAR γ and its target genes. We also found that HMG-CoA reductase (HMGCR) was downregulated in the mouse liver by EWGP. Our data suggest that EWGP lowers hyperlipidemia of C57BL/6 mice induced by high-fat diet via the inhibition of PPAR γ and HMGCR signaling.

8.
Artigo em Inglês | MEDLINE | ID: mdl-23320036

RESUMO

Obesity is a common nutritional disorder associated with type 2 diabetes, cardiovascular diseases, dyslipidemia, and certain cancers. In this study, we investigated the effects of Citrus ichangensis peel extract (CIE) in high-fat (HF) diet-induced obesity mice. Female C57BL/6 mice were fed a chow diet or an HF diet alone or supplemented with 1% w/w CIE for 8 weeks. We found that CIE treatment could lower blood glucose level and improve glucose tolerance. In the HF+CIE group, body weight gain, serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-c) levels, and liver triglyceride (TG) and TC concentrations were significantly (P < 0.05) decreased relative to those in the HF group. To elucidate the mechanism of CIE on the metabolism of glucose and lipid, related genes expression in liver were examined. In liver tissue, CIE significantly decreased the mRNA expression levels of peroxisome proliferator-activated receptor γ (PPARγ) and its target genes, such as fatty acid synthase (FAS) and acyl-CoA oxidase (ACO). Moreover, CIE also decreased the expression of liver X receptor (LXR) α and ß which are involved in lipid and glucose metabolism. These results suggest that CIE administration could alleviate obesity and related metabolic disorders in HF diet-induced obesity mice through the inhibition of PPARγ and LXR signaling.

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