Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.
Actins/genetics ; Animals ; Bone Morphogenetic Proteins/genetics ; *Cell Differentiation ; Chickens ; Cytomegalovirus/genetics ; Drug Delivery Systems ; Embryo/*cytology ; *Gene Therapy ; Green Fluorescent Proteins/genetics/*metabolism ; Humans ; Immunoenzyme Techniques ; Microscopy, Fluorescence ; Peptide Elongation Factor 1/genetics ; Pluripotent Stem Cells/*cytology ; Promoter Regions (Genetics)/*genetics ; Research Support, Non-U.S. Gov't ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription, Genetic/genetics

This study was purposed to investigate the expression of the growth-factor independence 1 (GFI1) in patients with leukemia and its clinical significance. Bone marrow mononuclear cells were obtained from 65 newly diagnosed leukemia patients including 24 acute myeloid leukemia (AML), 18 chronic myelogenous leukemia (CML), 6 acute lymphoblastic leukemia (ALL), 17 blast crisis of chronic myelogenous leukemia and 13 patients with iron deficiency anemia (IDA) were used as controls. The relative expression of gene gfi1 was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and taqman quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR). The results showed that gene expression of gene gfi1 in leukemia patients was obviously higher than that in controls and the difference was statistically significant (p < 0.01), in which the expression of gene gfi1 in newly diagnosed CML patients was higher than that in newly diagnosed AML, newly diagnosed ALL, CML-BCP patients and the difference was significant (p < 0.01). Expression of gene gfi1 in lymphocytic blast crisis of CML was higher than that in nonlymphocytic blast crisis of CML, and the difference was significant. It is concluded that gene gfi1 may play an important role in leukemia, especially in CML incidence and progression. The high level expression of gene gfi1 may be participate in the development of lymphoma.
DNA-Binding Proteins ; genetics ; Gene Expression ; Humans ; Leukemia ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription Factors ; genetics ; Transcription, Genetic ; genetics

OBJECTIVETo investigate the underlying tumor susceptibility mechanisms and reasons for the high risk of cancer in Fanconi anemia (FA).

METHODSGene Set Enrichment Analysis (GSEA) was performed to compare gene expression profiles between 21 FA patients' bone marrow (BM) mononuclear cell (BMNC) and 11 normal controls in cancer related gene sets from NCBI GEO database, then core enriched genes were identified by further investigation. Through enrichment analyzing biological processes of gene ontology sets and structural genomic gene sets between FA expression profiles and control, more details related with its tumor susceptibility had been revealed.

RESULTSCompared with normal control, gene expression in FA group had significant been enriched in resistance to Bcl-2 inhibitor gene set, fibroblast growth factors signalling pathways, insulin and insulin-like growth factors (IGF) signalling pathways induced cancer genesis gene sets. The high level of D4S234E, SST, FGFs, IGFs, FGFRs and IGFBP expression provided an initiate environment for tumorgenesis and drug resistance. There were significant differences in biogenesis extracellular molecules and cytomembrane structure organizations between FA and control. Genes with promoter regions around transcription start sites containing either motif RRCAGGTGNCV or CCTNTMAGA were enriched and those former genes match annotation for tumorgenic transcription factor 3 (TCF3).

CONCLUSIONSThe high tumor susceptibility of FA patients may be closely related with the dramatic changes in cancer related growth factors and hormones environment. This study provides new insights into tumor susceptibility mechanism in FA patients.

Basic Helix-Loop-Helix Transcription Factors ; genetics ; Case-Control Studies ; Fanconi Anemia ; genetics ; Female ; Gene Expression ; Genes, Neoplasm ; Genetic Predisposition to Disease ; Humans ; Insulin-Like Growth Factor Binding Proteins ; genetics ; Male ; Neoplasms ; genetics ; Promoter Regions, Genetic ; Signal Transduction ; genetics ; Somatomedins ; genetics ; Transcription Initiation Site ; Transcriptome

Transcription, Genetic

The recent mumps epidemic in South Korea has generated a large amount of public concern. This study has attempted to analyze molecular epidemiological changes of mumps virus circulating in Gwangju metropolitan area, South Korea. 953 throat swab samples were collected from patients with parotitis from May 2013 to July 2014. The majority (71.5%) of these cases have occurred in middle or high school students aged from 15 to 19 years. All samples were tested using a reverse transcription polymerase chain reaction (RT-PCR) that targets the short hydrophobic (SH) gene of the virus. Mumps virus SH gene was detected in 39.2% (374/953) of samples. And 82 RT-PCR products were randomly selected for nucleotide sequencing analysis. All of these sequences were determined as genotype I by phylogenetic analysis and showed the highest nucleic acid similarity (99%) with Dg1062/Korea/98 (GenBank accession no. AY309060). These results suggested that appearance of new genotype or genetic variation at the nucleotide level could be ruled out to evaluate main cause of recent mumps outbreak in Gwangju metropolitan area.
Genetic Variation ; Genotype ; Gwangju ; Humans ; Korea ; Molecular Epidemiology* ; Mumps ; Mumps virus* ; Parotitis ; Pharynx ; Polymerase Chain Reaction ; Reverse Transcription

OBJECTIVETo investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2.

METHODSWe established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45.

RESULTSWe found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45.

CONCLUSIONNF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.

Base Sequence ; Blood Proteins ; genetics ; DNA Primers ; GATA1 Transcription Factor ; physiology ; Gene Expression Regulation ; physiology ; Gene Silencing ; HeLa Cells ; Humans ; Methylation ; Molecular Chaperones ; genetics ; NF-E2 Transcription Factor, p45 Subunit ; physiology ; Promoter Regions, Genetic ; Reverse Transcriptase Polymerase Chain Reaction

Promoter prediction is a very important problem and is closely related to the main problems of bioinformatics such as the construction of gene regulatory networks and gene function annotation. In this context, we developed an integrated promoter prediction program using hybrid methods, PromoterWizard, which can be employed to detect the core promoter region and the transcription start site (TSS) in vertebrate genomic DNA sequences, an issue of obvious importance for genome annotation efforts. PromoterWizard consists of three main modules and two auxiliary modules. The three main modules include CDRM (Composite Dependency Reflecting Model) module, SVM (Support Vector Machine) module, and ICM (Interpolated Context Model) module. The two auxiliary modules are CpG Island Detector and GCPlot that may contribute to improving the predictive accuracy of the three main modules and facilitating human curator to decide on the final annotation.
Base Sequence ; Chimera ; Computational Biology ; CpG Islands ; Dependency (Psychology) ; Gene Regulatory Networks ; Genome ; Humans ; Promoter Regions, Genetic ; Transcription Initiation Site ; Vertebrates

CpG-island margins and non-island-CpG sites round the transcription start sites of CpG-island-positive and -negative genes are methylated to various degrees in a tissue-specific manner. These methylation-variable CpG sites were analyzed to delineate a relationship between the methylation and transcription of the tissue-specific genes. The level of tissue-specific transcription was estimated by counting the number of the total transcripts in the SAGE (serial analysis of gene expression) database. The methylation status of 12 CpG-island margins and 21 non-island CpG sites near the key tissue-specific genes was examined in pluripotent stromal cells obtained from fat and bone marrow samples as well as in lineage-committed cells from marrow bulk, stomach, colon, breast, and thyroid samples. Of the 33 CpG sites examined, 10 non-island-CpG sites, but none of the CpG-island margins were undermethylated concurrent with tissue-specific expression of their nearby genes. The net methylation of the 33 CpG sites and the net amount of non-island-CpG gene transcripts were high in stomach tissues and low in stromal cells. The present findings suggest that the methylation of the non-island-CpG sites is inversely associated with the expression of the nearby genes, and the concert effect of transitional-CpG methylation is linearly associated with the stomach-specific genes lacking CpG-islands.
Adipose Tissue/cytology ; Adolescent ; Adult ; Adult Stem Cells/cytology/*metabolism ; Aged ; CpG Islands/*genetics ; *DNA Methylation ; Female ; Gene Expression Profiling ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Stomach/cytology/*metabolism ; Stromal Cells/metabolism ; Transcription Initiation Site ; Transcription, Genetic

To investigate whether there are NUP98-HOXA, NUP98-HOXB, NUP98-HOXC, NUP98-HOXD fusion genes in leukemia patients in Xinjiang, cellular total RNA was extracted from the bone marrow mononuclear cells, the formaldehyde-agarose gel electrophoresis was used to judge whether RNA was intact, the 17 RT-PCR primers were designed to amplify the predicted fusion junctions and 412 bp GAPDH was used as an internal control, NUP98-HOXA fusion genes were amplified by nested-PCR following reverse transcription. One-step PCR was performed to amplify the other predicted fusion genes. The results showed that RNA was proved to be intact and expression of GAPDH was found in every sample. However, no predicted fusion transcripts were detected in leukemia patients. In conclusion, no NUP98-HOX fusion genes were detected in the samples from Xinjiang.
Adult ; Bone Marrow Cells ; metabolism ; Female ; Humans ; Leukemia ; blood ; genetics ; Leukocytes, Mononuclear ; metabolism ; Male ; Oncogene Proteins, Fusion ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Transcription, Genetic

OBJECTIVETo validate the conversion factor (CF) for the conversion of BCR-ABL (P210) transcript levels to the international scale in chronic myeloid leukemia (CML).

METHODSIn 2012, the international reference laboratory in Adelaide, Australia (IMVS) sent two batches of RNA samples, 30 samples per batch, to Peking University People's Hospital (PKUPH). By comparing BCRABL (P210) transcript levels reported by the two laboratories, CF of PKUPH was calculated and validated by IMVS. In 2013, PKUPH prepared the exchange samples for validation of CF of 9 hospitals who have calculated CFs before. The fresh BCR-ABL (P210) (+) cells were serially diluted by BCR-ABL (P210) (-) cells to prepare 22 kinds of samples with different BCR-ABL transcript levels, each kind had 10 parallel samples. Trizol reagent was added in each tube. Ten hospitals tested BCR-ABL transcript levels of one set of 22 samples. Agreement between BCR-ABL transcript levels of each laboratory and PKUPH was assessed by the Bland-Altman method.

RESULTSPKUPH successfully validated its CF with bias 1.1 fold and 95% limits of agreement between -4.7 and 4.9 fold. Of 9 hospitals whose validation performed by sample exchanges with PKUPH, 6 hospitals successfully validated their CF with bias ≤±1.4 fold and 95% limits of agreement within ±6 fold.

CONCLUSIONValidation of CF examined the stability of the detection of BCR-ABL (P210) transcript levels, which was necessary for the valid conversion of BCR-ABL (P210) transcript levels to the international scale in CML.

Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; standards ; Transcription, Genetic