Genetic updating and marks of cellular lines

2020 ◽  
Vol 22 (3) ◽  
pp. 168-175
Author(s):  
A. V. Moskalev ◽  
B. Yu. Gumilevskiy ◽  
A. V. Apchel ◽  
V. N. Tsygan
Keyword(s):  
Stem Cells ◽  
Deoxyribonucleic Acid ◽  
Target Genes ◽  
Viral Vector ◽  
Bacteriophage P1 ◽  
Biotechnology Research ◽  
New Genes ◽  
Delivery Vector ◽  
Cutting Out ◽  
Lentivirus Vectors

Abstract. Despite great advances in the biology of stem cells, there are still many dark spots. Genetic modification techniques, which can be used to track the lines of different cells, primarily stem cells, help to solve this problem. Various methods of biotechnology research are considered, allowing to evaluate the options of introducing new genes into cells and even whole organisms, as well as methods of controlling their expression in time and space, their activation, differentiation and decrease in functional activity, expression of several target genes. Options with multi-cystron vectors encoding several proteins are described. Options for introducing genes using plasmids, electroportation of their disadvantages and advantages are characterized. The most promising and the safest is a retroviral vector using lentivirus vectors capable of generating additional copies of itself, which is very important in the field of biotechnology security. A line of packing cells, usually 293T cells, is used to produce a viral vector. Prospects for the use of adenovirus and adenoassociated vectors are characterized. The achievement of modern biotechnology methods is the system of short palindrome repetitions located in groups, which is a unique tool for genome editing. At the heart of this system is the process of cutting out sequences of deoxyribonucleic acid, which are permanent and which are supported by cells regardless of subsequent divisions or changes in condition. The system allows geneticists and medical researchers to edit parts of the genome by removing, adding or modifying successive sites of deoxyribonucleic acid. An important problem with biotechnology methods is how to control the expression of transgenes. Today, it is quite effective to control expression with a factor present in the gene delivery vector itself and which is only active in a certain type of cell. Endonuclease bacteriophage P1 is used to regulate transgene expression, which cuts deoxyribonucleic acid only at specific sites. This system is introduced in both eukaryotic and prokaryotic systems.

scholarly journals BCOR Internal Tandem Duplication Expression in Neural Stem Cells Promotes Growth, Invasion, and Expression of PRC2 Targets

2021 ◽  
Vol 22 (8) ◽  
pp. 3913
Author(s):  
Satoshi Nakata ◽  
Ming Yuan ◽  
Jeffrey A. Rubens ◽  
Ulf D. Kahlert ◽  
Jarek Maciaczyk ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Tandem Duplication ◽  
Target Genes ◽  
Cellular Growth ◽  
Central Nervous System Tumor ◽  
Internal Tandem Duplication ◽  
Invasion And Migration ◽  
Human Neural Stem Cells ◽  
And Migration

Central nervous system tumor with BCL6-corepressor internal tandem duplication (CNS-BCOR ITD) is a malignant entity characterized by recurrent alterations in exon 15 encoding the essential binding domain for the polycomb repressive complex (PRC). In contrast to deletion or truncating mutations seen in other tumors, BCOR expression is upregulated in CNS-BCOR ITD, and a distinct oncogenic mechanism has been suggested. However, the effects of this change on the biology of neuroepithelial cells is poorly understood. In this study, we introduced either wildtype BCOR or BCOR-ITD into human and murine neural stem cells and analyzed them with quantitative RT-PCR and RNA-sequencing, as well as growth, clonogenicity, and invasion assays. In human cells, BCOR-ITD promoted derepression of PRC2-target genes compared to wildtype BCOR. A similar effect was found in clinical specimens from previous studies. However, no growth advantage was seen in the human neural stem cells expressing BCOR-ITD, and long-term models could not be established. In the murine cells, both wildtype BCOR and BCOR-ITD overexpression affected cellular differentiation and histone methylation, but only BCOR-ITD increased cellular growth, invasion, and migration. BCOR-ITD overexpression drives transcriptional changes, possibly due to altered PRC function, and contributes to the oncogenic transformation of neural precursors.

scholarly journals TrkA Interacts with and Phosphorylates STAT3 to Enhance Gene Transcription and Promote Breast Cancer Stem Cells in Triple-Negative and HER2-Enriched Breast Cancers

Cancers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2340
Author(s):  
Angelina T. Regua ◽  
Noah R. Aguayo ◽  
Sara Abu Jalboush ◽  
Daniel L. Doheny ◽  
Sara G. Manore ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Gene Transcription ◽  
Target Genes ◽  
Triple Negative ◽  
Breast Cancer Stem Cells ◽  
Breast Cancers ◽  
Co Activation ◽  
Stat3 Phosphorylation

JAK2–STAT3 and TrkA signaling pathways have been separately implicated in aggressive breast cancers; however, whether they are co-activated or undergo functional interaction has not been thoroughly investigated. Herein we report, for the first time that STAT3 and TrkA are significantly co-overexpressed and co-activated in triple-negative breast cancer (TNBC) and HER2-enriched breast cancer, as shown by immunohistochemical staining and data mining. Through immunofluorescence staining–confocal microscopy and immunoprecipitation–Western blotting, we found that TrkA and STAT3 co-localize and physically interact in the cytoplasm, and the interaction is dependent on STAT3-Y705 phosphorylation. TrkA–STAT3 interaction leads to STAT3 phosphorylation at Y705 by TrkA in breast cancer cells and cell-free kinase assays, indicating that STAT3 is a novel substrate of TrkA. β-NGF-mediated TrkA activation induces TrkA–STAT3 interaction, STAT3 nuclear transport and transcriptional activity, and the expression of STAT3 target genes, SOX2 and MYC. The co-activation of both pathways promotes breast cancer stem cells. Finally, we found that TNBC and HER2-enriched breast cancer with JAK2–STAT3 and TrkA co-activation are positively associated with poor overall metastasis-free and organ-specific metastasis-free survival. Collectively, our study uncovered that TrkA is a novel activating kinase of STAT3, and their co-activation enhances gene transcription and promotes breast cancer stem cells in TNBC and HER2-enriched breast cancer.

scholarly journals Influence of Egr-1 in Cardiac Tissue-Derived Mesenchymal Stem Cells in Response to Glucose Variations

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Daniela Bastianelli ◽  
Camilla Siciliano ◽  
Rosa Puca ◽  
Andrea Coccia ◽  
Colin Murdoch ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Target Genes ◽  
Cardiac Tissue ◽  
Early Response ◽  
Induced Response ◽  
Protein Levels ◽  
Short Period ◽  
Phenotype Analysis

Mesenchymal stem cells (MSCs) represent a promising cell population for cell therapy and regenerative medicine applications. However, how variations in glucose are perceived by MSC pool is still unclear. Since, glucose metabolism is cell type and tissue dependent, this must be considered when MSCs are derived from alternative sources such as the heart. The zinc finger transcription factor Egr-1 is an important early response gene, likely to play a key role in the glucose-induced response. Our aim was to investigate how short-term changes inin vitroglucose concentrations affect multipotent cardiac tissue-derived MSCs (cMSCs) in a mouse model of Egr-1 KO (Egr-1−/−). Results showed that loss of Egr-1 does not significantly influence cMSC proliferation. In contrast, responses to glucose variations were observed in wt but not in Egr-1−/−cMSCs by clonogenic assay. Phenotype analysis by RT-PCR showed that cMSCs Egr-1−/−lost the ability to regulate the glucose transporters GLUT-1 and GLUT-4 and, as expected, the Egr-1 target genes VEGF, TGFβ-1, and p300. Acetylated protein levels of H3 histone were impaired in Egr-1−/−compared to wt cMSCs. We propose that Egr-1 acts as immediate glucose biological sensor in cMSCs after a short period of stimuli, likely inducing epigenetic modifications.

scholarly journals miR-16-2* Interferes with WNT5A to Regulate Osteogenesis of Mesenchymal Stem Cells

2018 ◽  
Vol 51 (3) ◽  
pp. 1087-1102 ◽  
Author(s):  
Lijun Duan ◽  
He Zhao ◽  
Yang Xiong ◽  
Xiangsheng Tang ◽  
Yongdong Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Target Genes ◽  
Fragility Fractures ◽  
Inhibitory Effect ◽  
Lymphocytic Leukemia ◽  
Qrt Pcr ◽  
Wnt Signal

Background/Aims: Osteoporosis is a bone metabolic disease characterized by a systemic impairment of bone mass, which results in increased propensity of fragility fractures. A reduction in the differentiation of MSCs into osteoblasts contributes to the impaired bone formation observed in osteoporosis. Mesenchymal stem cells (MSCs) are induced to differentiate into preosteoblasts, which are regulated by the signaling cascades initiated by the various signals, including miRNAs. miR-16-2* is a newly discovered miRNA that participates in diagnosis and prognosis of hepatocellular carcinoma, cervical cancer and chronic lymphocytic leukemia. However, the effect of miR-16-2* on the regulation of osteoblast differentiation and the mechanism responsible are still unclear. Here we discuss the contribution of miR-16-2* to osteoporosis, osteoblast differentiation and mineralization. Methods: The expression pattern of miR-16-2* during osteogenesis or in osteoporosis bone samples was validated by quantitative real-time PCR (qRT-PCR). The human bone marrow mesenchymal stem cells (hBMSCs) were induced to differentiate into osteoblasts by osteogenic induced medium containing dexamethasone, ascorbate-2-phosphat, beta-glycerophosphate and vitamin-D3. The target genes of miR-16-2* were predicted by TargetScan and PicTar. The mRNA and protein levels of osteogenic key markers were detected using qRT-PCR or western blot respectively. The WNT signal activity was analyzed by TOP/FOP reporter assay. Results: The expression of miR-16-2* in patient bone tissue with osteoporosis was negatively correlated with bone formation related genes. During osteoblast differentiation process, the expression of miR-16-2* was significantly decreased. Upregulation of miR-16-2* in hBMSCs impaired the osteogenic differentiation while the downregulation of miR-16-2* increased this process. Upregulation the expression of miR-16-2* could also block the WNT signal pathway by directly target WNT5A. Furthermore, knockdown of miR-16-2* could promote the activation of RUNX2, possibly by lifting the inhibitory effect of miR-16-2* on WNT pathway. Conclusion: Taken together, we report a novel biological role of miR-16-2* in osteogenesis through regulating WNT5A response for the first time. Our data support the potential utilization of miRNA-based therapies in regenerative medicine.

Genome-wide analysis of familial dysautonomia and kinetin target genes with patient olfactory ecto-mesenchymal stem cells

2012 ◽  
Vol 33 (3) ◽  
pp. 530-540 ◽  
Author(s):  
Nathalie Boone ◽  
Aurélie Bergon ◽  
Béatrice Loriod ◽  
Arnaud Devèze ◽  
Catherine Nguyen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Target Genes ◽  
Familial Dysautonomia ◽  
Genome Wide Analysis ◽  
Genome Wide

scholarly journals New Insights in the Sugarcane Transcriptome Responding to Drought Stress as Revealed by Supersage

2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
Éderson Akio Kido ◽  
José Ribamar Costa Ferreira Neto ◽  
Roberta Lane de Oliveira Silva ◽  
Valesca Pandolfi ◽  
Ana Carolina Ribeiro Guimarães ◽  
...  
Keyword(s):  
Drought Stress ◽  
Target Genes ◽  
Present Report ◽  
Transcriptome Profiling ◽  
Water Deficit Stress ◽  
New Genes ◽  
Drought Tolerant ◽  
Blastn Analysis ◽  
Root Tissues ◽  
Solexa Sequencing Technology

In the scope of the present work, four SuperSAGE libraries have been generated, using bulked root tissues from four drought-tolerant accessions as compared with four bulked sensitive genotypes, aiming to generate a panel of differentially expressed stress-responsive genes. Both groups were submitted to 24 hours of water deficit stress. The SuperSAGE libraries produced 8,787,315 tags (26 bp) that, after exclusion of singlets, allowed the identification of 205,975 unitags. Most relevant BlastN matches comprised 567,420 tags, regarding 75,404 unitags with 164,860 different ESTs. To optimize the annotation efficiency, the Gene Ontology (GO) categorization was carried out for 186,191 ESTs (BlastN against Uniprot-SwissProt), permitting the categorization of 118,208 ESTs (63.5%). In an attempt to elect a group of the best tags to be validated by RTqPCR, the GO categorization of the tag-related ESTs allowed thein silicoidentification of 213 upregulated unitags responding basically to abiotic stresses, from which 145 presented no hits after BlastN analysis, probably concerning new genes still uncovered in previous studies. The present report analyzes the sugarcane transcriptome under drought stress, using a combination of high-throughput transcriptome profiling by SuperSAGE with the Solexa sequencing technology, allowing the identification of potential target genes during the stress response.

scholarly journals Hypoxia-inducible factor 1α induces osteo/odontoblast differentiation of human dental pulp stem cells via Wnt/β-catenin transcriptional cofactor BCL9

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Shion Orikasa ◽  
Nobuyuki Kawashima ◽  
Kento Tazawa ◽  
Kentaro Hashimoto ◽  
Keisuke Sunada-Nara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dental Pulp ◽  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
Dental Pulp Stem Cells ◽  
Pulp Tissue ◽  
Odontoblast Differentiation ◽  
Hypoxia Inducible Factor 1Α ◽  
Hypoxic Conditions

AbstractAccelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that hypoxia due to vascular severance may induce osteo/odontoblast differentiation of dental pulp stem cells (DPSCs). This study examined the role of B-cell CLL/lymphoma 9 (BCL9), which is downstream of hypoxia-inducible factor 1α (HIF1α) and a Wnt/β-catenin transcriptional cofactor, in the osteo/odontoblastic differentiation of human DPSCs (hDPSCs) under hypoxic conditions. hDPSCs were isolated from extracted healthy wisdom teeth. Hypoxic conditions and HIF1α overexpression induced significant upregulation of mRNAs for osteo/odontoblast markers (RUNX2, ALP, OC), BCL9, and Wnt/β-catenin signaling target genes (AXIN2, TCF1) in hDPSCs. Overexpression and suppression of BCL9 in hDPSCs up- and downregulated, respectively, the mRNAs for AXIN2, TCF1, and the osteo/odontoblast markers. Hypoxic-cultured mouse pulp tissue explants showed the promotion of HIF1α, BCL9, and β-catenin expression and BCL9-β-catenin co-localization. In addition, BCL9 formed a complex with β-catenin in hDPSCs in vitro. This study demonstrated that hypoxia/HIF1α-induced osteo/odontoblast differentiation of hDPSCs was partially dependent on Wnt/β-catenin signaling, where BCL9 acted as a key mediator between HIF1α and Wnt/β-catenin signaling. These findings may reveal part of the mechanisms of dental pulp mineralization after traumatic dental injury.

scholarly journals Wnt signaling recruits KIF2A to the spindle to ensure chromosome congression and alignment during mitosis

2021 ◽  
Vol 118 (34) ◽  
pp. e2108145118
Author(s):  
Anja Bufe ◽  
Ana García del Arco ◽  
Magdalena Hennecke ◽  
Anchel de Jaime-Soguero ◽  
Matthias Ostermaier ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Division ◽  
Wnt Signaling ◽  
Pluripotent Stem Cells ◽  
Target Genes ◽  
Genome Maintenance ◽  
Spindle Poles ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
Canonical Wnt

Canonical Wnt signaling plays critical roles in development and tissue renewal by regulating β-catenin target genes. Recent evidence showed that β-catenin–independent Wnt signaling is also required for faithful execution of mitosis. However, the targets and specific functions of mitotic Wnt signaling still remain uncharacterized. Using phosphoproteomics, we identified that Wnt signaling regulates the microtubule depolymerase KIF2A during mitosis. We found that Dishevelled recruits KIF2A via its N-terminal and motor domains, which is further promoted upon LRP6 signalosome formation during cell division. We show that Wnt signaling modulates KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle. Accordingly, inhibition of basal Wnt signaling leads to chromosome misalignment in somatic cells and pluripotent stem cells. We propose that Wnt signaling monitors KIF2A activity at the spindle poles during mitosis to ensure timely chromosome alignment. Our findings highlight a function of Wnt signaling during cell division, which could have important implications for genome maintenance, notably in stem cells.

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