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Optimization of reporter gene assay:several factors influencing detection of promoter activity

Chinese Medical Journal 2007年第11期120卷 965-969页

作者： XUE Li-xiang WENG Mo ZHANG Zong-yu TONG Tan-jun Department or Biochemistry and Molecular Biology Health Science Center Peking University Beijing 100083 China

Background promoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role and has been used widely in the promoter assay. Methods Human embryonic lung fibroblast cells （2BS）, HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined vadous factors that affect promoter activity determination, such as the selection of the reporter genes and internal references, the dose and the type of the vectors carrying the transcription factors, the host cells and the instruments. Results The sensitivity of the luciferase assay was much higher than that of enhanced green fluorescence protein （EGFP）. Moreover, promoter activity is increased in a dose-related manner only in certain ranges outside of which the results may be reversed and the promoter activity is related to the expression vector which is carrying the cDNA. Otherwise, the length of the promoter, internal references and the host cell can also influence the promoter activity. Conclusions To detect the promoter activity accurately, a few factors including dose, vector, length and host cell which influence reporter gene assay aforementioned should be considered.

关键词： promoter regions activity assay luciferases green fluorescent proteins biological factors

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Identification of Klebsiella pneumoniae strains harboring inactive extended-spectrum beta-lactamase antibiotic-resistance genes

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Chinese Medical Journal 2014年第17期127卷 3051-3057页

作者： Xu Li Zhai Yao Lyu Yuan Wang Qi An Shuchang Chen Jichao Chen Yusheng Liu Lin Li Jiabin Gao Zhancheng Department of Respiratory and Critical Care Medicine Peking University People's Hospital Beijing 100044 China Institute of Clinical Pharmacology Peking University First Hospital Peking University Beijing 100034 China Department of Respiratory Medicine the Second Affiliated Hospital to Dalian Medical University Dalian Liaoning 116023 China Department of Respiratory Medicine First Hospital of Tsinghua University Beijing 100016 China Department of-Respiratory Medicine the Central Hospital of China Aerospace Corporation Beijing 100049 China Department of Respiratory Medicine Fujian Provincial Hospital Fuzhou Fujian 350001 China Department of Infectious Diseases First Affiliated Hospital of Anhui Medical University Hefei Anhui 230022 China

Background The extended-spectrum beta-lactamase （ESBL）-producing Klebsiella pneumoniae has increasingly become a major contributor to nosocomial infections and can exhibit multiple antibiotic *** studies have focused on the resistance genes in ESBL-producing strains,and the resistance-associated genetic environment of non-ESBL-producing strains has been ignored until ***,we investigated the occurrence and characteristics of non-ESBL-producing ***,which potentially carries unexpressed resistance *** *** strains were collected from five medical institutions in China from February 2010 to August *** VITEK-2 ESBL detection system was used as a primary screen to identify the ESBL-producing phenotype,and the three primary types of ESBL-associated genes （CTX,SHV,and TEM） were detected by polymerase chain reaction （PCR） to confirm the strains presenting with a non-ESBL-producing *** expression in the non-ESBL-producing strains was further screened by reverse-transcription PCR （RT-PCR） to validate their transcriptional *** Out of 224 clinically isolated antibiotic-sensitive *** strains with a non-ESBL-producing phenotype,5 （2.2％） were identified to carry inactivated ESBL blaSHV genes with intact upstream promoter regions and resistance gene ***,three of the five antibiotic-sensitive *** strains containing ESBL blaSHV genes still exhibited mRNA transcription of blasHv,while the other two exhibited no mRNA *** These findings suggest that inactivated ESBL genes exist in non-ESBL-producing antibiotic-sensitive *** strains,which have the potential to transform the strain into an ESBL phenotype if an inappropriate application or overdose of antibiotics is implemented during clinical management.

关键词： Klebsiella pneumoniae extended-spectrum beta-lactamase gene silencing beta-lactam resistance promoter regions

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Genetic and epigenetic changes associated with cholangiocarcinoma:From DNA methylation to microRNAs

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World Journal of Gastroenterology 2007年第48期13卷 6465-6469页

作者： Monique Stutes Steven Tran Sharon DeMorrow Division of Research and Education Scott and White Hospital Temple Texas Department of Medicine Texas A&M Health Science Center College of Medicine Temple Texas

Cholangiocarcinomas are malignant epithelial liver tumors arising from the intra- and extra-hepatic bile ducts. Little is known about the molecular development of this disease, and very few effective treatment options are available. Thus, prognosis is poor. Genetic and epigenetic changes play an integral role in the neoplastic transformation of human cells to their malignant counterparts. This review summarizes some of the more prevalent genetic alterations （by microRNA expression） and epigenetic changes （hypermethylation of specific gene promoters） that are thought to contribute to the carcinogenic process in cholancliocarcinoma.

关键词： promoter regions CpG islands Tumor suppressor Oncogene Inflammation

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