AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astr...AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells （HSC）. In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS： The GFAP-lacZ transgene was transfected into various cell lines （HSC, hepatocyte, and other nonHSC cell types）. The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS： The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION： In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.展开更多
The development of genetically modified crops requires new promoters and regulatory regions to achieve high gene ex- pression and/or tissue-specific expression patterns in plants. To obtain promoter sequences of plant...The development of genetically modified crops requires new promoters and regulatory regions to achieve high gene ex- pression and/or tissue-specific expression patterns in plants. To obtain promoter sequences of plants with new properties, we analyzed the expression traits of the cotton （Gossypium hirsutum） translation elongation factor 1A gene family. The results showed that the GhEF1A8 gene is highly expressed in different organs of cotton plants, and showed much higher transcript levels in stems and leaves. Its promoter （GhEFIA1.7） and the 5＂ untranslated region （5＂ UTR）, comprising a regulatory region named PGhEFIA8, were isolated from cotton and studied in stably transformed tobacco plants. The regulatory region sequences were fused to the 13-glucuronidase （GUS） reporter gene to characterize its expression pattern in tobacco. Histochemical and fiuorometric GUS activity assays demonstrated that PGhEF1A8 could direct GUS gene expression in all tissues and organs in transgenic tobacco, including leaves, stems, flowers, and roots. The level of GUS activity in the leaves and stems was significantly higher than in cauliflower mosaic virus （CaMV） 35S promoter：：GUS plants, but as same as CaMV 35S promoter：：GUS plants in flower and root tissues. GUS expression levels decreased 2-10-fold when the 5＂ UTR was absent from PGhEF1A8. Deletion analysis of the PGhEFIA8 sequence showed that the region -647 to -323 might possess negative elements that repress transgene expression in tobacco plants. The results suggested that the GhEFIA8 regulation region may represent a practical choice to direct high-level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering.展开更多
OBJECTIVE: To clone and study the polymorphism within interleukin-4 (IL-4) proximal promoter of asthmatic children. METHODS: The IL-4 proximal promoter segments were amplified and selected by polymerase chain reaction...OBJECTIVE: To clone and study the polymorphism within interleukin-4 (IL-4) proximal promoter of asthmatic children. METHODS: The IL-4 proximal promoter segments were amplified and selected by polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) with genomic DNA from ten healthy children and forty patients with dominantly allergic familial histories as templates. The selected PCR segments were cloned into recombinant plasmids pIL-4-Jx2. The PCR inserts were sequenced by dideoxy chain termination method. RESULTS: Seven aberrant bands were found in SSCP analysis from forty asthmatic patients. The sequencing results showed that four variant sites were found within or adjacent to the known IL-4 regulatory element. A C to A transversion located at -229 position was just within the positive regulatory element-I (PRE-I) in one patient. A C to T transition adjacent to the negative regulatory element-II (NRE-II) and an extra G adjacent to TATA box were found in two patients. A five base nucleotide deletion was found near signal transducers and activators of transcription-6 responsive element (STAT-6 RE) in one patient. CONCLUSION: There were polymorphisms within the IL-4 proximal promoter of allergic asthmatic patients and these polymorphisms might result in aberrant expression of IL-4 gene and asthma.展开更多
目的研究重庆市汉族人群Klotho基因启动子区域G-395A单核苷酸多态性的分布，探讨该多态性住点与动脉硬化的相关性。方法232倒健康体检者均进行臂踝脉搏波传导速度测定，并记录动脉硬化的传统危险因素，根据测定结果分为动脉硬化组（130...目的研究重庆市汉族人群Klotho基因启动子区域G-395A单核苷酸多态性的分布，探讨该多态性住点与动脉硬化的相关性。方法232倒健康体检者均进行臂踝脉搏波传导速度测定，并记录动脉硬化的传统危险因素，根据测定结果分为动脉硬化组（130例）和对照组（102例）。应用TaqMan探针等位基因特异性杂交分析法对Klotho基因G-395A多态性位点进行分析。结果G-395A多态性住点共检测出GG、GA、AA3种基因型，频率分别为60．3％、34．1％和5．6％，符合Hardy-Weinberg平衡。动脉硬化组-395A等位基因的频率显著低于对照组（33．1％ VS 48.0％，P=0．022）。logistic回归分析，调整传统危险因素后-395A与动脉硬化呈负相关（P=0．042，OR=0．537，95％CI：0．295-0．977）。结论Klotho基因-395A等位基因可能是动脉硬化的遗传学保护因素。展开更多
目的:探讨DNA结合抑制因子4(inhibitor of DNA binding 4,ID4)基因核心启动子区甲基化及其蛋白表达与儿童急性白血病(acute leukemia,AL)的关系。方法:采用甲基化特异性聚合酶链反应(methylation-specific polymerase chain reaction,MS...目的:探讨DNA结合抑制因子4(inhibitor of DNA binding 4,ID4)基因核心启动子区甲基化及其蛋白表达与儿童急性白血病(acute leukemia,AL)的关系。方法:采用甲基化特异性聚合酶链反应(methylation-specific polymerase chain reaction,MS-PCR)对32例初发白血病患儿、34例同期非肿瘤疾病儿童(对照组)及白血病细胞株Jurkat和Molt4进行ID4基因启动子区甲基化状况分析,并进行测序。RT-PCR检测ID4 mRNA的表达,免疫组织化学法检测ID4蛋白的表达。结果:ID4基因启动子区在Jurkat和Molt4细胞中均呈完全甲基化状态,而初发急性淋巴细胞及非淋巴细胞白血病患儿中完全甲基化率显著高于对照组患儿(81.0%vs17.6%,P<0.001;63.6%vs17.6%,P<0.005)。测序分析显示在完全甲基化状态的ID4基因核心启动子区所有CpG岛均呈甲基化状态。呈完全甲基化状态的白血病患儿和Jurkat、Molt4细胞中均无ID4 mRNA表达,而白血病组ID4蛋白表达显著低于对照组(P<0.001)。结论:儿童白血病及某些白血病细胞株存在ID4基因核心启动子区高程度的甲基化。完全的甲基化状态可使ID4基因表达沉默,ID4蛋白表达缺失。展开更多
基金Supported by the Biomedical Research Councilthe Institute of Bioengineering and Nanotechnology,the Republic of Singapore
文摘AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells （HSC）. In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS： The GFAP-lacZ transgene was transfected into various cell lines （HSC, hepatocyte, and other nonHSC cell types）. The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS： The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION： In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.
基金supported by the New Genetically Modified Organisms Varieties Cultivation Project, China (2014ZX08005-004)
文摘The development of genetically modified crops requires new promoters and regulatory regions to achieve high gene ex- pression and/or tissue-specific expression patterns in plants. To obtain promoter sequences of plants with new properties, we analyzed the expression traits of the cotton （Gossypium hirsutum） translation elongation factor 1A gene family. The results showed that the GhEF1A8 gene is highly expressed in different organs of cotton plants, and showed much higher transcript levels in stems and leaves. Its promoter （GhEFIA1.7） and the 5＂ untranslated region （5＂ UTR）, comprising a regulatory region named PGhEFIA8, were isolated from cotton and studied in stably transformed tobacco plants. The regulatory region sequences were fused to the 13-glucuronidase （GUS） reporter gene to characterize its expression pattern in tobacco. Histochemical and fiuorometric GUS activity assays demonstrated that PGhEF1A8 could direct GUS gene expression in all tissues and organs in transgenic tobacco, including leaves, stems, flowers, and roots. The level of GUS activity in the leaves and stems was significantly higher than in cauliflower mosaic virus （CaMV） 35S promoter：：GUS plants, but as same as CaMV 35S promoter：：GUS plants in flower and root tissues. GUS expression levels decreased 2-10-fold when the 5＂ UTR was absent from PGhEF1A8. Deletion analysis of the PGhEFIA8 sequence showed that the region -647 to -323 might possess negative elements that repress transgene expression in tobacco plants. The results suggested that the GhEFIA8 regulation region may represent a practical choice to direct high-level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering.
文摘OBJECTIVE: To clone and study the polymorphism within interleukin-4 (IL-4) proximal promoter of asthmatic children. METHODS: The IL-4 proximal promoter segments were amplified and selected by polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) with genomic DNA from ten healthy children and forty patients with dominantly allergic familial histories as templates. The selected PCR segments were cloned into recombinant plasmids pIL-4-Jx2. The PCR inserts were sequenced by dideoxy chain termination method. RESULTS: Seven aberrant bands were found in SSCP analysis from forty asthmatic patients. The sequencing results showed that four variant sites were found within or adjacent to the known IL-4 regulatory element. A C to A transversion located at -229 position was just within the positive regulatory element-I (PRE-I) in one patient. A C to T transition adjacent to the negative regulatory element-II (NRE-II) and an extra G adjacent to TATA box were found in two patients. A five base nucleotide deletion was found near signal transducers and activators of transcription-6 responsive element (STAT-6 RE) in one patient. CONCLUSION: There were polymorphisms within the IL-4 proximal promoter of allergic asthmatic patients and these polymorphisms might result in aberrant expression of IL-4 gene and asthma.
文摘目的研究重庆市汉族人群Klotho基因启动子区域G-395A单核苷酸多态性的分布，探讨该多态性住点与动脉硬化的相关性。方法232倒健康体检者均进行臂踝脉搏波传导速度测定，并记录动脉硬化的传统危险因素，根据测定结果分为动脉硬化组（130例）和对照组（102例）。应用TaqMan探针等位基因特异性杂交分析法对Klotho基因G-395A多态性位点进行分析。结果G-395A多态性住点共检测出GG、GA、AA3种基因型，频率分别为60．3％、34．1％和5．6％，符合Hardy-Weinberg平衡。动脉硬化组-395A等位基因的频率显著低于对照组（33．1％ VS 48.0％，P=0．022）。logistic回归分析，调整传统危险因素后-395A与动脉硬化呈负相关（P=0．042，OR=0．537，95％CI：0．295-0．977）。结论Klotho基因-395A等位基因可能是动脉硬化的遗传学保护因素。
