scholarly journals Visualizing Gene Expression in Real-Time

2005 ◽  
Vol 13 (3) ◽  
pp. 3-7
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Real Time ◽  
Temporal Resolution ◽  
Protein Sequence ◽  
Living Cells ◽  
Entire Process ◽  
Gene Chips ◽  
A Cell ◽  
Dna Template

Gene expression has been visualized for a few decades, but in static forms such as blots and gene chips. Susan Janicki, Toshiro Tsukamoto, Sim one Salghetti, William Tansey, Ravi Sachidanandam, Kannanganattu Prasanth, Thomas Ried, Yaron Shav-Tal, Edouard Bertrand, Robert Singer, and David Spector have recently designed a cell line in which gene expression can be observed with stunningly accurate spatial and temporal resolution. Gene expression is a cascade of events beginning with transcription of RNA from the DNA template and ending with translation into a protein sequence. Janicki et al. were able to visualize the entire process at the levels of DNA, RNA, and proteins in living cells!

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.

scholarly journals Development of Caudal Fin Cell Line from Hill Trout Barilius Bendelisis (Hamilton, 1807) for Cytotoxicity and Transfection Studies

2021 ◽  
Vol 79 (1) ◽  
pp. 15-24
Author(s):  
Murali Sanjeev Kumar ◽  
Pankaj Soni ◽  
Ravindra Kumar ◽  
Neha Singh ◽  
Shreya Srivastava ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Mitochondrial Gene ◽  
Toxicity Screening ◽  
Green Fluorescence ◽  
Caudal Fin ◽  
A Cell ◽  
Barilius Bendelisis ◽  
Early Toxicity ◽  
N Vector

Abstract A cell line named BBdF-1, established from the caudal fin of hill stream fish Barilius bendelisis, has been subcultured for more than 52 passages and is being maintained in L-15 media containing 20% FBS. Species origin of the cell line was confirmed using amplification of partial region of 16S and COI mitochondrial gene sequences. The optimum temperature for growth of BBdF-1 cell line was found to be 28°C. Karyotyping revealed diploid chromosome number as 50. Cells exhibited strong binding for cytokeratin marker and thus were found to be epithelial-like. Strong green fluorescence was observed in BBdF-1 cells transfected with phrGFP-II-N vector, indicating its suitability for utilization in gene expression and manipulation studies. Successful assessment of cytotoxicity of two heavy metals, viz. mercury and chromium, was performed. The cell line can serve as a useful resource material for early toxicity screening of pesticides/pollutant and gene expression.

scholarly journals In Vivo and In Vitro Analysis of Baculovirusie-2 Mutants

1999 ◽  
Vol 73 (3) ◽  
pp. 2460-2468 ◽  
Author(s):  
Elena A. Prikhod’ko ◽  
Albert Lu ◽  
Joyce A. Wilson ◽  
Lois K. Miller
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Late Gene ◽  
Wild Type ◽  
Late Gene Expression ◽  
Specific Effects ◽  
Viral Promoters ◽  
Occlusion Bodies ◽  
A Cell ◽  
Budded Virus

ABSTRACT Upon transient expression in cell culture, the ie-2gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) displays three functions: transactivation of viral promoters, direct or indirect stimulation of virus origin-specific DNA replication, and arrest of the cell cycle. The ability of IE2 to trans stimulate DNA replication and coupled late gene expression is observed in a cell line derived fromSpodoptera frugiperda but not in a cell line derived fromTrichoplusia ni. This finding suggested that IE-2 may exert cell line-specific or host-specific effects. To examine the role of ie-2 in the context of infection and its possible influence on the host range, we constructed recombinants of AcMNPV containing deletions of different functional regions within ie-2 and characterized them in cell lines and larvae of S. frugiperda and T. ni. Theie-2 mutant viruses exhibited delays in viral DNA synthesis, late gene expression, budded virus production, and occlusion body formation in SF-21 cells but not in TN-5B1-4 cells. In TN-5B1-4 cells, the ie-2 mutants produced more budded virus and fewer occlusion bodies but the infection proceeded without delay. Examination of the effects of ie-2 and the respective mutants on immediate-early viral promoters in transient expression assays revealed striking differences in the relative levels of expression and differences in responses to ie-2 and its mutant forms in different cell lines. In T. ni and S. frugiperda larvae, the infectivities of the occluded form ofie-2 mutant viruses by the normal oral route of infection was 100- and 1,000-fold lower, respectively, than that of wild-type AcMNPV. The reduction in oral infectivity was traced to the absence of virions within the occlusion bodies. The infectivity of the budded form of ie-2 mutants by hemocoelic injection was similar to that of wild-type virus in both species. Thus,ie-2 mutants are viable but exhibit cell line-specific effects on temporal regulation of the infection process. Due to its effect on virion occlusion, mutants of IE-2 were essentially noninfectious by the normal route of infection in both species tested. However, since budded viruses exhibited normal infectivity upon hemocoelic injection, we conclude that ie-2 does not affect host range per se. The possibility that IE-2 exerts tissue-specific effects has not been ruled out.

Molecular Beacons for Sensitive Gene Detection in Living Cells

2003 ◽  
Author(s):  
Andrew Tsurkas ◽  
Gang Bao
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Molecular Beacon ◽  
Point Mutations ◽  
Living Cells ◽  
Molecular Beacons ◽  
High Signal ◽  
Gene Detection ◽  
Real Time Imaging ◽  
Background Ratio

Real-time imaging of gene expression in living cells has the potential to significantly impact clinical and laboratory studies of cancer, including cancer diagnosis and analysis. Molecular beacons (MBs) provide a simple and promising tool for the detection of target mRNA as tumor markers due to their high signal-to-background ratio, and their improved specificity in detecting point mutations. However, the harsh intracellular environment does limit the sensitivity of MB-based gene detection. Specifically, MBs bound to target mRNAs cannot be distinguished from those degraded by nucleases, or opened due to non-specific interactions. To overcome this difficulty, we have developed a novel dual FRET molecular beacons approach in which a pair of molecular beacons, one with a donor fluorophore and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of false-positive events. We have performed systematic in-solution and cellular studies of dual FRET molecular beacon and demonstrated that this new approach allows for real-time imaging of gene expression in living cells.

65 SCRIPTAID CORRECTS GENE EXPRESSION OF A FEW ABERRANTLY REPROGRAMMED TRANSCRIPTS IN NUCLEAR TRANSFER PIG BLASTOCYST STAGE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 138
Author(s):  
K. M. Whitworth ◽  
J. Zhao ◽  
L. D. Spate ◽  
R. S. Prather
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Real Time ◽  
Histone Deacetylase ◽  
Real Time Pcr ◽  
Nuclear Transfer ◽  
Transcriptional Profiling ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Hdac Activity

Scriptaid is a histone deacetylase inhibitor (HDACi) that can increase cloning efficiency. The objective of this study was to identify aberrantly reprogrammed transcripts by performing transcriptional profiling between in vivo (IVV), nuclear transfer (NT) blastocyst stage embryos and the donor cell line (cells). This was followed by measuring HDAC activity (Epigentek) in zygotes and by real-time PCR on a selected subset of genes at the blastocyst stage to determine if Scriptaid treatment (NTS) corrected the aberrant gene expression. NTS embryos were treated with 500 nM Scriptaid for 14 h after activation. NT and NTS embryos were transferred into gilts on Day 0 or 1 of oestrus and collected 6 days later by uterine flush. IVV samples were collected on Day 8 of gestation. 3 pools of 10 to 15 embryos and cells were collected for each treatment and analysed twice. For transcriptional profiling, total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA), amplified by using an Ovation Ribo-SPIA linear amplification kit (Nugen), labelled with Cy5 and compared to reference labelled with Cy3. Lowess normalization and analysis was performed in Genespring 7.3.1. ANOVA was performed with the Benjamini and Hochberg False Discovery Rate. Transcripts that were different between IVV and NT (P ≤ 0.20) and significantly different from the donor cell line (P ≤ 0.05) were classified as being aberrantly reprogrammed. This comparison resulted in 119 under- and 60 over-compensated transcripts. Functional annotation classification was performed in DAVID and identified under-compensated pathways (oxidative phosphorylation and protein biosynthesis) and over-compensated pathways (chromatin packaging/remodelling and protein complex assembly). Fourteen transcripts were chosen for real-time PCR validation and evaluation of the effect of Scriptaid. Relative gene expression was compared between IVV, NT, NTS, and cells by the comparative Ct method with SYBR Green Supermix (Bio-Rad) and statistical analysis was performed in SAS 9.1 (SAS Institute Inc., Cary, NC, USA) by using a least significant difference test (P ≤ 0.05). NTS embryos had 3 transcripts returning to the same level as IVV (H3F3A, CAPG, and SEPT7). The level of the majority of the transcripts (8/14) was not affected by NTS treatment, e.g. histone deacetylase SIRT1 and H1 histone, member 0 (H1F0). However, Scriptaid treatment caused COX5A to be further over compensated in NTS with expression levels higher than IVV and NT. 2 transcripts had expression levels that were lower in NTS compared to both IVV and NT including GPD1L and EIF3E. Scriptaid treatment significantly affected gene expression in 6 of the 14 transcripts evaluated. Scriptaid treatment of the reconstructed zygotes did not affect the majority of the transcripts when measured at the blastocyst stage. HDAC activity was significantly reduced in NTS compared to NT 1-cell stage embryos (P ≤ 0.038). While Scriptaid reduced HDAC activity, it returned only a few genes to normal IVV levels. This project was supported in part by the USDA NRI (2006-35203-17282) and Food for the 21st Century.

Thymoquinone Effects on Cell Viability, Apoptosis and VEGF-A Gene Expression Level in AGS(CRL-1739) Cell Line

2019 ◽  
Vol 19 (6) ◽  
pp. 820-826 ◽  
Author(s):  
Mohsen Rashid ◽  
Forough Sanjarin ◽  
Farzaneh Sabouni
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Cell Line ◽  
Real Time ◽  
Mtt Assay ◽  
Nigella Sativa ◽  
Angiogenic Factor ◽  
Cancer Drug ◽  
Anti Cancer ◽  
Ags Cell Line

Background: Cancer is one of the most fatal diseases across the world and it was reported that 90% of cancer fatality depends on its angiogenesis potential. Black seed or Nigella sativa L. is a medicinal plant native to southwest Asia. N. sativa has been used for medicinal purposes for centuries and predominantly has bioactive components like Thymoquinone, which is used as a candidate for anti-cancer and anti-angiogenesis drugs. Methods: Callus was induced from leaf tissue, after that alcoholic extracts were prepared from three-month-old calluses. Thymoquinone content was measured by HPLC methods. AGS cell line was cultured and treated with standard Thymoquinone and extracts from callus. Then, cell proliferation, expression of angiogenic factor (VEGF-A gene), and apoptosis test were done by MTT assay, real-time PCR and Annexin-v kit, respectively. Results: HPLC found the maximum amount of Thymoquinone in the extract of leaf calluses, which were grown in the dark. MTT assay revealed that particular doses of extracts reduced cell proliferation. Real-time and Fluorescence- Activated Cell Sorting (FACS) results demonstrated that standard Thymoquinone and callus extracts down-regulated the VEGF-A gene expression, and all three induced apoptosis in the AGS cell line. Conclusion: It has been shown that TQ has pro-apoptotic and anti-metastatic effects on stomach cancer cell line, and these properties can introduce it as an anti-cancer drug in the near future.

Non-centrosomal microtubule formation and measurement of minus end microtubule dynamics in A498 cells

1997 ◽  
Vol 110 (19) ◽  
pp. 2391-2401 ◽  
Author(s):  
A.M. Yvon ◽  
P. Wadsworth
Keyword(s):  
Cell Line ◽  
De Novo ◽  
Human Kidney ◽  
Light Level ◽  
Pause Duration ◽  
Living Cells ◽  
Live Cells ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
A Cell

Experiments performed on a cell line (A498) derived from a human kidney carcinoma revealed non-centrosomal microtubules in the peripheral lamella of many cells. These short microtubules were observed in glutaraldehyde-fixed cells by indirect immunofluorescence, and in live cells injected with rhodamine-labeled tubulin. The non-centrosomal microtubules were observed to form de novo in living cells, and their complete disassembly was also observed. Low-light-level fluorescence microscopy, coupled to imaging software, was utilized to record and measure the dynamic behavior of both ends of the non-centrosomal microtubules in these cells. For each, the plus end was differentiated from the minus end using the ratio of their transition frequencies and by measuring total assembly at each end. For comparative purposes, dynamics of the plus ends of centrosomally nucleated microtubules were also analyzed in this cell line. Our data reveal several striking differences between the plus and minus ends. The average pause duration was nearly 4-fold higher at the minus ends; the percentage of time spent in pause was 92% at the minus ends, compared to 55% at plus ends. Dynamicity was decreased 4-fold at the minus ends, and the average number of events per minute was reduced from 7.0 at the plus end to 1.5 at the minus ends. The minus ends also showed a 6-fold decrease in frequency of catastrophe over the plus ends. These data demonstrate that in living cells, microtubules can form at sites distant from the perinuclear microtubule organizing center, and once formed, non-centrosomal microtubules can persist for relatively long periods.

scholarly journals Partial silencing of fucosyltransferase 8 gene expression inhibits proliferation of Ishikawa cells, a cell line of endometrial cancer

2020 ◽  
Vol 22 ◽  
pp. 100740
Author(s):  
Hana Shimoyama ◽  
Toshiaki K. Shibata ◽  
Masahiko Ito ◽  
Tomoaki Oda ◽  
Toshiya Itoh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endometrial Cancer ◽  
Cell Line ◽  
Ishikawa Cells ◽  
A Cell

scholarly journals The Establishment of Bortezomib Resistant Myeloma Cell Line KM3/BTZ and Explore the Resistance Mechanism

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5226-5226 ◽  
Author(s):  
Luqun Wang ◽  
Qiong Liu ◽  
Hao Li ◽  
Li Lizhen ◽  
Xin Wang
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Resistance Index ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Rt Pcr ◽  
A Cell ◽  
Family Gene ◽  
Mdr 1

Abstract Background: Multiple myeloma (MM) is an incurable B-cell malignancy resulting in significant morbidity and mortality, the incidence of second place in hematological malignancies. The proteasome inhibitor bortezomib inhibits IkappaB degradation, prevents NF-kappaB activation, and induces apoptosis in MM cells, has become first-line drug to MM. Despite its promising activity in traditional chemoresistant myeloma patients, however, some patients are resistant or become refractory to bortezomib. Chauhan D et al reported that Blockade of Hsp27 overcomes bortezomib resistance in lymphoma cells (Chauhan D et al., Cancer Re, 2003). However, bortezomib resistance mechanisms in MM remain controversial, the molecular basis of this reduced responsiveness is currently not fully understood. Objective: To establish a bortezomib-resistant cell line KM3 /BTZ of MM, then search for genes related to bortezomib resistance phenotype by analyzing the differential gene expression patterns with cDNA microarray,and explain the mechanism of bortezomib-resistant multiple myeloma. Methods: Bortezomib-resistant KM3/BTZ cell line was generated by increasing concentration of bortezomib to induce KM3 cells in vitro. MTT assay was employed to detect the cytotoxic effect on KM3 and KM3/BTZ cells. The cells apoptosis were analyzed by flow cytometry. We detect the gene expression profile changes between KM3/BTZ and its parent cell line KM3 cells by CDNA microarray. Combining of molecular annotation system MAS3.0 software and detailed analysis of documented resistance genes were used to analyze the data. Specific differentially-expressed genes were chosen for further verification using real-time RT-PCR. The expression of multi-drug resistance 1(MDR-1) gene mRNA was determined by RT-PCR. Results: The KM3/BTZ cell line was established successfully. Bortezomib resistance index values were significantly higher in KM3/BTZ (IC50 =351.2±3.51ng /ml) when compared to KM3 cells (IC50 = 17.8±1.03ng /ml),and the resistance index is 19.7 (P< 0.05). Through gene expression profiling filtering, compared with KM3,770 significantly differently expressed genes were screened out in the KM3/BTZ cell line, of which up and down regulated genes were 287 and 383 respectively, in which heat shock protein family gene HSPB2 was significantly up-regulated (Ratio=14.7455). Downregulated gene list in KM3/BTZ cell line included transcriptional regulators like ZNF family proteins (Tab 1). Upregulated genes in KM3/BTZ cell line included signal transduction related genes like MS4A family protein (Tab 1) also transcriptional regulators like ZNF family proteins (Tab 1). The expression trend of eight genes for further verification was almost consistent with the microarray, excepting gene of JUN (Fig 1). The expression of MDR-1 mRNA was not observed in either resistant or parental cells (Fig 2). Conclusion: We have successfully established bortezomib-resistant cell line KM3/BTZ. MDR-1 was not involved in bortezomib-resistant multiple myeloma cells.HSPB2, ZNF and MS4A family genes are probably related to bortezomib resistance in KM3/BTZ cells. Our results suggest that they may be new targets to overcome resistance to bortezomib in patients with MM. Table 1. ZNF and MS4A family gene expression in KM3/BTZ and KM3 cells Gene B signal* B detection* A signal* A detection* BvsA signal log ratio* BvsA change* ZNF711 85.08 P 206.16 P 0.41269 D ZNF92 6.75 P 14.57 P 0.46328 D ZNF704 10.7 A 26.78 P 0.39955 D ZNF492 7.08 P 3.48 P 2.03448 I ZNF532 87.93 P 43.54 A 2.01952 I ZNF594 10.25 P 4.63 P 2.21382 I ZNF789 269.79 P 97.3 P 2.77276 I ZNF506 330.36 P 149.48 P 2.21006 I MS4A3 1721.58 P 145.3 P 11.84845 I MS4A4A 2688.23 P 100.8 P 26.66895 I MS4A6A 141.96 P 56.47 M 2.513901 I MS4A1 49.02 P 53.11 P 0.92299 NC P: High expression; A: Low expression; M:Marginally D: Decrease; I: Increase; NC: No change *: A:KM3; B: KM3/BTZ Figure 1 Figure 1. Correlations of gene expression changes between oligonueleotide Microarray and qRT-PCR, showing change of selected gene in KM3/BTZ compared with KM3.The Y-axis indicates the log2 transformed ratio of mRNA expression. Figure 2 Figure 2. The expression level of MDR-1 mRNA in KM3 and KM3/BTZ and K562/A cell lines. M: DNA Marker; 1-2: K562/A cell line; 3-4: KM3cell line; 5-6: KM3/BTZ cell line Disclosures No relevant conflicts of interest to declare.

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