scholarly journals Derivation and Characterization of a ES-Like Cell Line from Indian CatfishHeteropneustes fossilisBlastulas

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Anindya S. Barman ◽  
Kuldeep K. Lal ◽  
Gaurav Rathore ◽  
Vindhya Mohindra ◽  
Rajeev K. Singh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Es Cells ◽  
Embryonic Cells ◽  
Dna Profile ◽  
Expression Studies ◽  
C Subunit ◽  
Gfp Reporter Gene ◽  
A Cell ◽  
Gene Expression Studies

A cell line designated as HFB-ES was established from blastula stage embryos ofH. fossilis(Singhi). The embryonic cells were harvested and maintained in Leibovitz’s medium supplemented with 15% fetal bovine serum. The cell line had been subcultured for more than 90 passages in a period of 24 months. HFB-ES cells were able to grow at temperatures between 25 and 35°C with an optimum temperature of 28°C. The growth rate of HFB-ES was proportional to FBS concentration, with optimum growth seen at 15% FBS concentration. The originality of the cell line was confirmed by sequencing of cytochrome oxidase c subunit I (COI), cytochrome b gene, and microsatellite DNA profile. Results of chromosome complements of HFB showed normal karyo-morphology with 56 (2n) diploid number of chromosomes after 40 passages which indicated that the developed cell line is chromosomally stable. The pluripotency of HFB was demonstrated by alkaline phosphatase activity and Oct-4 gene expression. Expression of GFP reporter gene was successful in HFB-ES. These results indicated that HFB-ES could be utilized for future gene expression studies.

Establishment and characterization of a piscean PCF cell line for toxicity and gene expression studies as in vitro model

2014 ◽  
Vol 46 (3) ◽  
pp. 206-212 ◽  
Author(s):  
M. Goswami ◽  
B.S. Sharma ◽  
Kamalendra Yadav ◽  
S.N. Bahuguna ◽  
W.S. Lakra
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
In Vitro Model ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Vitro Model

Serial Analysis of Gene Expression Revealed Consistent Downregulation of More Than 100 Genes in Hodgkin Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4288-4288
Author(s):  
Joost Kluiver ◽  
Ines Schwering ◽  
Debora de Jong ◽  
Ralf Küppers ◽  
Sibrand Poppema ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Hodgkin Lymphoma ◽  
Tumor Cells ◽  
Cell Lines ◽  
Pcr Analysis ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
B Lineage ◽  
Lineage Specific Genes

Abstract Hodgkin Lymphoma (HL) is unique in its cellular composition, consisting of a minority of tumor cells in an inflammatory background. The tumor cells (Reed-Sternberg cells, RS cells) are thought to be derived from germinal center B cells (centroblasts, CB) that have escaped apoptosis during antigen selection. Global gene expression studies in HL have been hampered by the fact that RS cells only represent less than 1% of the cells in the tumor tissue. Only a few large-scale gene expression studies are available, mostly using cell lines derived from patients with classical HL. To get more insight into the gene expression profile of RS cells we applied the Serial Analysis of Gene Expression Technique (SAGE) on several HL cell lines. SAGE was performed on the cell lines L428 and L1236 (classical cell lines, cHL), DEV (Nodular Lymphocyte Predominant cell line, NLP) and on CB as normal counterparts. In total, about 100.000 tags were sequenced (Table 1). After normalization and exclusion of tags present only once, 3.635 genes were retained and used for further analyses. A five-fold difference in tag frequency between the HL cell lines and CB was used to select for up- or downregulated genes. Although many genes were upregulated in every individual HL cell line compared to CB, only few genes were consistently upregulated. In contrast, comparison of the downregulated genes revealed that 125 were downregulated in all three HL cell lines compared to CB (Table 2). Tags corresponding to Fascin, TARC/CCL17 and BIC, known to be highly expressed in HL indeed were observed in the SAGE libraries. Among the genes downregulated in L428 a loss of B lineage-specific genes was observed confirming the previous findings in L1236. Interestingly our SAGE results suggest that the NLP cell line DEV has an intermediate loss of B-lineage specific genes compared to CB. To gain more insight in the biology, differentially expressed genes were grouped according to gene ontology’s (based on biological process or molecular function) and pathways. We have now selected more than 70 genes that will be subjected to quantitative RT-PCR analysis to confirm our SAGE results and to study their expression in tumor cell enriched HL samples. In conclusion: Downregulation of gene expression seems to be important in the pathogenesis of HL since we observed a consistent downregulation of more than 100 genes in HL compared to CB. Table 1. Overview of Hodgkin lymphoma SAGE libraries Table 2. Number of up- and downregulated genes in Hodgkin lymphoma compared to centroblasts Library DEV L428 L1236 CB HL type NLP cHL cHL Centroblasts Tags 16316 20990 30623 29787 Genes 4703 5602 10561 11095 L428 L1236 cHL DEV cHL & NLP HL L428 and L1236 are derived from classical Hodgkin lyphoma (cHL) cases, DEV is derived from a case of Nodular Predominant Hodgkin lymhoma (NLP HL) Upregulated 396 116 14 330 7 Downregulated 329 169 141 309 125

Gene Regulation during the Erythrocytic Differentiation of Embryonic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4255-4255
Author(s):  
Ewa Carrier ◽  
Shermila Kausal ◽  
Anand S. Srivastava
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Growth Factors ◽  
Es Cells ◽  
Embryonic Stem ◽  
Globin Genes ◽  
Rt Pcr ◽  
Expression Studies ◽  
Gene Expression Studies

Abstract We have studied the in vitro differentiation of murine embryonic stem cells (ES cells) towards erythropoiesis and expression of genes during this process. It has been reported that dexamethasone directs ES cells towards erythrocytic differentiation but the mechanism of gene regulation induced by dexamethasone is not well understood. We hypothesized that dexamethasone induces upregulation of erythropoietic genes such as GATA-1, FLK-1, EPO-R and directs ES cells towards erythropoietic differentiation. Murine ES cells (129 CCE) obtained from Dr. Nagy laboratory, Canada (Nagy et al., Histochem Cell Biol., 2001; 115:49–58) were subjected to the in vitro primary hematopoietic differentiation media containing methylcellulose, IMDM, IL -3, IL-6 and SCF (stem cell factor) without LIF (leukemia inhibitory factor) to promote embryoid body (EB) formation. Total RNA was collected on day 3, 5 and 9 EBs for gene expression studies using RT-PCR. On day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combination 1) SCF, EPO, dexamethasone, IGF, 2) SCF, IL-3, IL-6, TPO, 3) SCF IL-3, IL-6, TPO, EPO. Total RNA from day12 of secondary differentiated ES cells was collected to study cytokines and growth factors dependent erythrocytic differentiation and gene regulation, using RT-PCR. Our results demonstrate upregulation of Gata-1, Flk-1, HoxB-4, Epo-R and globin genes (α-globin, BH-1 globin, β-major globin, e -globin and z-globin) in the 9 days old EBs, whereas, RNA collected from 5 days old EBs showed expression of HoxB-4, e-globin, γ-globin, BH1-globin and FLK-1. Three days old EBs showed only HoxB-4 and FLK-1 gene expression and lack of expression of globin genes, indicating that erythtropoiesis-specific genes activate later. Gene expression studies of RNA collected from secondary differentiated ES cells and media containing dexamethasone showed downregulation of GATA-3 and upregulation of GATA-1, Flk-1 and Epo-R in comparison to the two other cytokines and growth factors media combination. These results confirm our hypothesis that dexamethasome induces erythropoiesis by down regulating GATA -3 and upregulating erythropoietic-related genes such as GATA-1, Flk-1 and Epo-R. The morphological characteristics of cells after secondary differentiation showed enhanced production of erythrocytic precursors in dexamethasone containing media, which corresponded with molecular studies. Further studies will address the role of wnt/β-catenin and E-cadherin in this process.

Establishment and Characterization of a New Muscle Cell Line of Zebrafish (Danio rerio) as an In Vitro Model for Gene Expression Studies

2016 ◽  
Vol 27 (3) ◽  
pp. 166-173 ◽  
Author(s):  
Amit Kumar ◽  
Neha Singh ◽  
Mukunda Goswami ◽  
J. K. Srivastava ◽  
Akhilesh K. Mishra ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Danio Rerio ◽  
In Vitro Model ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Vitro Model ◽  
Muscle Cell Line

scholarly journals Evaluation of Normalization Strategies Used in Real-time Quantitative PCR Experiments in HepaRG Cell Line Studies

2014 ◽  
Vol 60 (3) ◽  
pp. 451-454 ◽  
Author(s):  
Liesbeth Ceelen ◽  
Jurgen De Craene ◽  
Ward De Spiegelaere
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Heparg Cell ◽  
Expression Studies ◽  
Heparg Cell Line ◽  
Heparg Cells ◽  
Gene Expression Studies

Abstract BACKGROUND The HepaRG cell line is widely used as an alternative for primary human hepatocytes for numerous applications, including drug screening, and is progressively gaining importance as a human-relevant cell source. Consequently, increasing numbers of experiments are being performed with this cell line, including real-time quantitative PCR (RT-qPCR) experiments for gene expression studies. CONTENT When RT-qPCR experiments are performed, results are reliable only when attention is paid to several critical aspects, including a proper normalization strategy. Therefore, in 2011 we determined the most optimal reference genes for gene expression studies in the HepaRG cell system, according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. This study additionally provided clear evidence that the use of a single reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S18 (RPS18), or actin, beta (ACTB)] was insufficient for normalization in HepaRG cells. Our screening of relevant studies published after our study suggested that the findings of our study were completely ignored. SUMMARY In none of the 24 reviewed studies was a proper normalization method used. Only 1 reference gene was included for normalization in 21 out of the 24 reported studies we screened, with RPS18 and GAPDH used most frequently, followed by hypoxanthine phosphoribosyltransferase 1 (HPRT1), glutathione synthetase (GSS) (hGus), β-2 microglobin (B2M), and acidic ribosomal phosphoprotein P0 (36B4). For 2 studies the use of multiple reference genes (2 and 3) was reported, but these had not been prevalidated for expression stability in HepaRG cells. In 1 study, there was no evidence that any reference gene had been used. Current RT-qPCR gene expression studies in HepaRG cells are being performed without adequate consideration or evaluation of reference genes. Such studies can yield erroneous and biologically irrelevant results.

scholarly journals Comprehensive Integration of Genome-Wide Association and Gene Expression Studies Reveals Novel Gene Signatures and Potential Therapeutic Targets for Helicobacter pylori-Induced Gastric Disease

2021 ◽  
Vol 12 ◽  
Author(s):  
Mohamed Tarek Badr ◽  
Mohamed Omar ◽  
Georg Häcker
Keyword(s):  
Gene Expression ◽  
Helicobacter Pylori ◽  
Cell Line ◽  
Gene Signature ◽  
Genome Wide Association ◽  
Expression Studies ◽  
Genome Wide ◽  
Human Pathology ◽  
Gene Expression Studies ◽  
H Pylori

Helicobacter pylori is a gram-negative bacterium that colonizes the human gastric mucosa and can lead to gastric inflammation, ulcers, and stomach cancer. Due to the increase in H. pylori antimicrobial resistance new methods to identify the molecular mechanisms of H. pylori-induced pathology are urgently needed. Here we utilized a computational biology approach, harnessing genome-wide association and gene expression studies to identify genes and pathways determining disease development. We mined gene expression data related to H. pylori-infection and its complications from publicly available databases to identify four human datasets as discovery datasets and used two different multi-cohort analysis pipelines to define a H. pylori-induced gene signature. An initial Helicobacter-signature was curated using the MetaIntegrator pipeline and validated in cell line model datasets. With this approach we identified cell line models that best match gene regulation in human pathology. A second analysis pipeline through NetworkAnalyst was used to refine our initial signature. This approach defined a 55-gene signature that is stably deregulated in disease conditions. The 55-gene signature was validated in datasets from human gastric adenocarcinomas and could separate tumor from normal tissue. As only a small number of H. pylori patients develop cancer, this gene-signature must interact with other host and environmental factors to initiate tumorigenesis. We tested for possible interactions between our curated gene signature and host genomic background mutations and polymorphisms by integrating genome-wide association studies (GWAS) and known oncogenes. We analyzed public databases to identify genes harboring single nucleotide polymorphisms (SNPs) associated with gastric pathologies and driver genes in gastric cancers. Using this approach, we identified 37 genes from GWA studies and 61 oncogenes, which were used with our 55-gene signature to map gene-gene interaction networks. In conclusion, our analysis defines a unique gene signature driven by H. pylori-infection at early phases and that remains relevant through different stages of pathology up to gastric cancer, a stage where H. pylori itself is rarely detectable. Furthermore, this signature elucidates many factors of host gene and pathway regulation in infection and can be used as a target for drug repurposing and testing of infection models suitability to investigate human infection.

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