scholarly journals Novel miRNA Sponges to Specifically Modulate Gene Expression in Colon Cancer Cells

2019 ◽  
Author(s):  
Alejandro Aguilera ◽  
Gloria Perazzoli ◽  
Laura Cabeza ◽  
Cristina Mesa ◽  
Francisco Quiñonero ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Vector System ◽  
Egfp Expression ◽  
Expression Levels ◽  
Selective Blockade ◽  
Novel Mirna ◽  
Mirna Sponges ◽  
Green Fluorescent

miRNA sponges allow the selective blockade of a complete family of associated miRNAs which induce posttranscriptional gene silencing in its target through binding to 3´UTR mRNA. MiRNA-365 and miRNA-145 are down-regulated in colorectal cancer (CRC), but not in health tissues. Based on this, we constructed two vectors by inserting miRNA sponge (one for miRNA-365 and other for miRNA-145), and used EGFP (enhanced green fluorescent protein) as a 3′ UTR reporter gene to analyse the ability of each sponge to catch its respective miRNA. qPCR results corroborated that the expression levels of both miRNAs were lower in CRC cell lines than in normal colon cell line. Flow cytometry analysis revealed a decrease of the EGFP expression levels in the cell lines transfected with both sponges, being higher on the normal cell line while CRC cell lines presented a minimal decline. Also, this decrease was inversely proportional to the levels of expression of both miRNAs obtained by qPCR. These results were corroborated by fluorescence microscopy, showing a similar decrease fluorescence. We propose a new vector system to carry in a specific way the expression of genes to CRC cells without affecting healthy cells, preventing damage to healthy tissues.

Autophagic and Apoptotic Effects of Tyrosine Kinase Inhibitors in Myeloid Leukemia: Comparison of Three Generation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4915-4915
Author(s):  
Cagla Kayabasi ◽  
Cigir Biray Avci ◽  
Sunde Yilmaz Susluer ◽  
Tugce Balci ◽  
Yusuf Baran ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Control Group ◽  
Green Fluorescent

Abstract Abstract 4915 The aim of the study was to evaluate the differences in cytotoxicity, apoptosis and autophagy levels in myeloid leukemia cell lines treated with tyrosine kinase inhibitors compared to cell line resistant to imatinib and control group. Chronic myeloid leukemia model was created by using cell lines as K-562 cell line for Ph+ chronic myeloid leukemia model, HL-60 cell line for acute promyelocytic Ph- leukemia model. NCI-BL2171 normal cell line was used as a control group while K562/ima3 cell line was used as an imatinib resistant model. Imatinib (STI571), Dasatinib (BMS-354825), Ponatinib (AP24534) were used as tyrosine kinase inhibitors in this study. Cytotoxicity analysis was conducted by WST-1 analysis. Apoptotis was evaluated by AnnexinV-enhanced green fluorescent protein (EGFP) and by Mitoprobe JC-1 for Mitochondrial Potential Detection. Autophagy was analyzed by The Premo Autophagy Tb/GFP TR-FRET LC3B assay which measures autophagy in cells expressing green fluorescent protein (GFP)-tagged LC3B using a Tb-based TR-FRET immunoassay approach. By using IC50 doses of tyrosine kinase inhibitors, autophagic effect of these drugs on cell lines were examined at 24th hours. Cells not treated with the active substance or chloroquine were considered as control groups. Chloroquine-treated cells were used as positive control for autophagy. LC3B-II increase is an indicator of autophagic suppression. Cells treated with chloroquine were compared with cells treated with active substances and concentrations of BacMam that displayed the highest LC3B-II increase were selected. Autophagic suppression ratio of the drugs was evaluated among the control group. Cytotoxicity, apoptosis and autophagy analysis results were provided in Table. Compared to control group, 30 μM chloroquine repressed autophagy 1. 93, 1. 48, 2. 74 and 1. 54 fold in K562, HL-60, K562/ima3 and NCI-BL 2171 cells, respectively. In HL-60 cells while Imatinib represented 0. 77 fold autophagy, it repressed autophagy 1. 77 and 3. 49 fold in K562 and K562/ima3 cells respectively. Dasatinib repressed autophagy 2. 11, 1. 95 and 4. 62 fold and Ponatinib repressed autophagy 2. 09, 1. 60 and 9. 15 fold in K562, HL-60, K562/ima3 cells respectively. Imatinib, Dasatinib and Ponatinib did not repressed autophagy in NCI-BL 2171 cells. In conclusion, apoptosis and autophagy paradox was illuminated in myeloid leukemia cells via tyrosine kinase inhibitors and autophagy may be a new strategy for targeted therapy in myeloid leukemia after clarifying responsible genes and proteins in signal transduction pathways. Cytotoxicity Apoptosis Autophagy WST-1 IC50 (nM) Annexin V JC-1 Premo Autophagy Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib Ýmatinib Dasatinib Ponatinib K562 24th hour 1.70 3.65 3.05 3.07 1.37 1.35 1.43 1.77 2.11 2.09 48th hour 650.00 0.24 2.67 2.51 2.32 2.03 2.35 2.06 72nd hour 4.53 4.81 3.00 2.97 3.07 2.50 HL-60 24th hour 1.33 1.26 1.32 1.29 1.22 1.34 0.77 1.95 1.60 48th hour 18000.00 1.39 1.23 1.41 1.61 1.92 1.96 72nd hour 896.00 607.00 2.21 1.80 2.82 1.58 1.73 2.23 K562/ima3 24th hour 1.33 0.76 1.69 1.51 1.36 1.59 3.49 4.62 9.15 48th hour 18350.00 1830.00 9.87 1.80 1.94 2.03 2.82 1.22 1.40 72nd hour 1.34 1.44 1.41 2.61 1.40 1.56 NCI-BL 2171 24th hour 48.00 2.48 2.79 2.62 3.99 4.04 4.25 1.01 0.88 0.90 48th hour 274.00 30.00 4.11 4.33 4.15 5.05 2.75 3.11 72nd hour 6.14 6.04 6.03 8.27 3.71 3.95 Disclosures: No relevant conflicts of interest to declare.

scholarly journals Exogenous expression of Pit-1 in AtT-20 corticotropic cells induces endogenous growth hormone gene transcription

2002 ◽  
Vol 172 (3) ◽  
pp. 477-487 ◽  
Author(s):  
R Kurotani ◽  
S Yoshimura ◽  
Y Iwasaki ◽  
K Inoue ◽  
A Teramoto ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Green Fluorescent Protein Gene ◽  
Growth Hormone Gene ◽  
Pituitary Cells ◽  
Gh Gene ◽  
Exogenous Expression ◽  
Green Fluorescent

The pituitary-specific POU-homeodomain transcription factor, Pit-1, is known to regulate the expression of the GH gene in somatotropes, prolactin (PRL) in lactotropes, and TSH in thyrotropes. It is not normally expressed in corticotropes or gonadotropes. We addressed the question of whether exogenous Pit-1 was sufficient to induce ectopic transcription of the GH gene in the corticotropic cell line, AtT-20, or the gonadotropic cell line, alpha T3-1. A fusion gene composed of enhanced green fluorescent protein gene and human Pit-1 cDNA was transfected into AtT-20 and alpha T3-1 cells. The endogenous mouse GH mRNA was induced in three of nine AtT-20 cell lines and one of three alpha T3-1 cell lines containing the fusion gene. A small amount of GH protein was also detected in these cell lines. These data indicate that transfected Pit-1 is capable of inducing transcription of the GH gene in AtT-20 cells and alpha T3-1 cells. These data also suggest that synergistic co-factors might be required to transcribe the GH gene effectively for translation into GH protein. Furthermore, our findings support the hypothesis that the function of anterior pituitary cells is determined by the combinatorial action of specific transcription factors.

scholarly journals Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 632
Author(s):  
Yingyun Cai ◽  
Shuiqing Yu ◽  
Ying Fang ◽  
Laura Bollinger ◽  
Yanhua Li ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Hemorrhagic Fever ◽  
Simian Hemorrhagic Fever Virus ◽  
Hemorrhagic Fever Virus ◽  
Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

scholarly journals New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

2016 ◽  
Vol 82 (8) ◽  
pp. 2240-2246 ◽  
Author(s):  
Alex I. Kanno ◽  
Cibelly Goulart ◽  
Henrique K. Rofatto ◽  
Sergio C. Oliveira ◽  
Luciana C. C. Leite ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Expression Vectors ◽  
Content Type ◽  
Expression Levels ◽  
Wide Range ◽  
Mycobacteriophage L5 ◽  
Green Fluorescent ◽  
Selection Of

ABSTRACTThe expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

Novel features of intermediate filament dynamics revealed by green fluorescent protein chimeras

1998 ◽  
Vol 111 (13) ◽  
pp. 1767-1778 ◽  
Author(s):  
C.L. Ho ◽  
J.L. Martys ◽  
A. Mikhailov ◽  
G.G. Gundersen ◽  
R.K. Liem
Keyword(s):  
Green Fluorescent Protein ◽  
Dynamic Behavior ◽  
Cell Lines ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Fluorescent Protein ◽  
Nih3t3 Cells ◽  
Green Fluorescent ◽  
Filament Network ◽  
Perinuclear Area

In order to study the dynamic behavior of intermediate filament networks in living cells, we have prepared constructs fusing green fluorescent protein to intermediate filament proteins. Vimentin fused to green fluorescent protein labeled the endogenous intermediate filament network. We generated stable SW13 and NIH3T3 cell lines that express an enhanced green fluorescent protein fused to the N-terminus of full-length vimentin. We were able to observe the dynamic behavior of the intermediate filament network in these cells for periods as long as 4 hours (images acquired every 2 minutes). In both cell lines, the vimentin network constantly moves in a wavy manner. In the NIH3T3 cells, we observed extension of individual vimentin filaments at the edge of the cell. This movement is dependent on microtubules, since the addition of nocodazole stopped the extension of the intermediate filaments. Injection of anti-IFA causes the redistribution or ‘collapse’ of intermediate filaments. We injected anti-IFA antibodies into NIH3T3 cells stably expressing green fluorescent protein fused to vimentin and found that individual intermediate filaments move slowly towards the perinuclear area without obvious disassembly. These results demonstrate that individual intermediate filaments are translocated during the collapse, rather than undergoing disassembly-induced redistribution. Injections of tubulin antibodies disrupt the interactions between intermediate filaments and stable microtubules and cause the collapse of the vimentin network showing that these interactions play an important role in keeping the intermediate filament network extended. The nocodazole inhibition of intermediate filament extension and the anti-IFA microinjection experiments are consistent with a model in which intermediate filaments exhibit an extended distribution when tethered to microtubules, but are translocated to the perinuclear area when these connections are severed.

Asymmetrical targeting of type II Na-Picotransporters in renal and intestinal epithelial cell lines

2000 ◽  
Vol 278 (3) ◽  
pp. F361-F368 ◽  
Author(s):  
N. Hernando ◽  
S. Sheikh ◽  
Z. Karim-Jimenez ◽  
H. Galliker ◽  
J. Forgo ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Intestinal Epithelial ◽  
Type Ii ◽  
Opossum Kidney ◽  
Epithelial Cell Lines ◽  
Molecular Determinants ◽  
Cellular Context ◽  
Green Fluorescent

Targeting of newly synthesized transporters to either the apical or basolateral domains of polarized cells is crucial for the function of epithelia, such as in the renal proximal tubule or in the small intestine. Recently, different sodium-phosphate cotransporters have been identified. Type II cotransporters can be subdivided into two groups: type IIa and type IIb. Type IIa is predominantly expressed in renal proximal tubules, whereas type IIb is located on the intestinal and lung epithelia. To gain some insights into the polarized targeting of the type II cotransporters, we have transiently expressed type IIa and type IIb cotransporters in several epithelial cell lines: two lines derived from renal proximal cells (opossum kidney and LLC-PK1), one from renal distal cells (Madin-Darby canine kidney), and one from colonic epithelium (CaCo-2). We studied the expression of the transporters fused to the enhanced green fluorescent protein. Our data indicate that the polarized targeting is dependent on molecular determinants most probably located at the COOH terminus of the cotransporters as well as on the cellular context.

scholarly journals A new transgene mouse model using an extravesicular EGFP tag to elucidate the in vivo function of extracellular vesicles

2021 ◽  
Author(s):  
Mikkel Oernfeldt Noegaard ◽  
Lasse Bach Steffensen ◽  
Didde Hansen ◽  
Ernst-Martin Fuchtbauer ◽  
Morten Buch Engelund ◽  
...  
Keyword(s):  
Mouse Model ◽  
Extracellular Vesicles ◽  
Fluorescent Protein ◽  
Urine Samples ◽  
Egfp Expression ◽  
Reporter Mice ◽  
Plasma And Urine Samples ◽  
Co Precipitation ◽  
Green Fluorescent

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate. We therefore created an EV reporter using CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9-EGFP expression in cells did not affect EV size and concentration, but allowed for co-precipitation of EV markers TSG101 and ALIX from cell-conditioned medium by anti-GFP immunoprecipitation. We created a transgenic mouse where CD9-EGFP was inserted in the inverse orientation and double-floxed, ensuring Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (AMHC-Cre) and kidney epithelium (Pax8-Cre), respectively. The mice showed tissue-specific EGFP expression, and plasma and urine samples were used to immunoprecipitate EVs. CD9-EGFP EVs was detected in plasma samples from CMV-Cre/CD9-EGFP and AMHC-Cre/CD9-EGFP mice, but not in PAX8-Cre/CD9-EGFP mice. On the other hand, CD9-EGFP EVs were detected in urine samples from CMV-Cre/CD9-EGFP and PAX8-Cre/CD9-EGFP mice, but not AMHC-Cre/CD9-EGFP, indicating that plasma EVs are not filtered to the urine. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to study cell-specific EVs in vivo and gain new insight into their physiological and pathophysiological function.

Mammalian cell lines expressing functional RNA polymerase II tagged with the green fluorescent protein

2000 ◽  
Vol 113 (15) ◽  
pp. 2679-2683 ◽  
Author(s):  
K. Sugaya ◽  
M. Vigneron ◽  
P.R. Cook
Keyword(s):  
Green Fluorescent Protein ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Temperature Sensitive ◽  
Mammalian Cell Lines ◽  
Eukaryotic Genes ◽  
Green Fluorescent

RNA polymerase II is a multi-subunit enzyme responsible for transcription of most eukaryotic genes. It associates with other complexes to form enormous multifunctional ‘holoenzymes’ involved in splicing and polyadenylation. We wished to study these different complexes in living cells, so we generated cell lines expressing the largest, catalytic, subunit of the polymerase tagged with the green fluorescent protein. The tagged enzyme complements a deficiency in tsTM4 cells that have a temperature-sensitive mutation in the largest subunit. Some of the tagged subunit is incorporated into engaged transcription complexes like the wild-type protein; it both resists extraction with sarkosyl and is hyperphosphorylated at its C terminus. Remarkably, subunits bearing such a tag can be incorporated into the active enzyme, despite the size and complexity of the polymerizing complex. Therefore, these cells should prove useful in the analysis of the dynamics of transcription in living cells.

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