Analysis of gene expression of hESCs upon genetic modification with EBNA and S/MAR episomal vectors
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE142299 | GEO | 2021/12/05
REPOSITORIES: GEO
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Project description:Analysis of gene expression of hESCs upon genetic modification with EBNA and S/MAR episomal vectors
| PRJNA596482 | ENA
Project description:Methods: Autonomously replicating vectors represent a simple and versatile model system for genetic modifications, but their localisation in the nucleus is largely unknown. Using circular chromosome conformation capture we mapped genomic contact sites of S/MAR-based replicons in HeLa cells. The influence of cis-active sequences on genomic localisation was assessed using replicons containing either an insulator sequence or an intron Results: While the original and the insulator-containing replicons displayed distinct contact sites, the intron-containing replicon showed a rather broad genomic contact pattern. Our results indicate a preference for certain chromatin structures and a rather non-dynamic behaviour during mitosis. Independent of inserted cis-active elements established vector molecules reside preferentially within actively transcribed regions, especially within promoter sequences and transcription start sites. Conclusions: S/MAR-based episomal replicons have a limited number of preferential contact sites and seem to be fairly non-dynamic during mitosis. We show that cis-acting elements do have an impact on the chormosomal localisation of episomal replicons, even though the epigenetic signatue of these contact sites are similar. Independently of the inserted cis-acting element, these contact sites are preferentially located within actively transcribed regions, especially promoter sites. Knowledge of preferred contact sites of exogenous DNA, e.g. viral or non-viral episomes, contribute to our understanding of episome behaviour in the nucleus and can be used for vector improvement and guiding of DNA sequences to specific subnuclear sites.
2017-09-18 | GSE97858 | GEO
Project description:Methods: mRNA profiles of untransfected HeLa cells (wild-type; wt) were compared with mRNA profiles of HeLa cells stably maintaining an S/MAR-based episome. Results: We here report for the first time that episomally maintained S/MAR-based vectors do not alter gene expression profile of the host cell's genome. No global changes in gene expression in episome maintaining cells, compared to non-transfected cells could be observed. To identify differentially expressed genes, false discovery rate (FDR; q-value) cut off was set to 0.01. Significantly differentially expressed genes with q<0.01 and an absolute fold-change of 2 were not detected. For verification, we chose five genes with high fold-change and low q-values (q<0.05) and compared expression levels between untransfected HeLa and HeLa stably maintaining an S/MAR-based within three replicates in qPCR. Conclusions: S/MAR-based replicons used in this study do not code for viral proteins but tend to co-localise with promoter sequences and transcription start sites. Recent observations that cooperatively transcribed promoters can influence each other raise concerns that S/MAR-based replicons have the potential to alter endogenous gene expression. Therefore, we compared the transcriptome of untransfected HeLa cells with HeLa cells stably maintaining an S/MAR-based episome. Setting the FDR to <0.01, we found no significantly differentially expressed genes. This finding is of utmost importance for potential gene therapeutic application of S/MAR-based replicons.
2017-09-18 | GSE97725 | GEO
Project description:Two cell lines, BL41K3 and BJABK3, were chosen to perform a comprehensive screen for EBNA-2 target genes. Both cell lines are EBV negative B-cell lines and express a chimeric EBNA-2 protein fused to the hormone binding domain of the estrogen receptor (ER/EBNA-2). The cells are propagated in the absence of hormone 31,36. Estrogen stimulation activates ER/EBNA-2, which in turn activates well known EBNA-2 target genes like CD21. BL41K3 and BJABK3 were cultured in estrogen supplemented medium for 24h and gene expression profiles were established for unstimulated and stimulated cells. Keywords: Transcription factor target identification
2006-10-03 | GSE4525 | GEO
Project description:Non-integrating minimally sized Nano-S/MAR DNA vectors can be used to genetically modify dividing cells in place of integrating vectors such as lentivirus and sleeping beauty. They represent a unique genetic tool, which avoids vector-mediated genetic damage cells and the activation of innate immune responses. Previous work has shown that DNA vectors comprising the mammalian scaffold/matrix attachment region (S/MAR) element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations which reduce their stability and can provoke cellular toxicity. Here, we present a new system, the Nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to their minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells by using Nano-S/MARs to stably restore the tumour suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that this class of DNA vector can be used to persistently modify a wide range of cells, providing sustained high levels of transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.
2021-12-12 | GSE142117 | GEO
Project description:Non-integrating minimally sized Nano-S/MAR DNA vectors can be used to genetically modify dividing cells in place of integrating vectors such as lentivirus and sleeping beauty. They represent a unique genetic tool, which avoids vector-mediated genetic damage cells and the activation of innate immune responses. Previous work has shown that DNA vectors comprising the mammalian scaffold/matrix attachment region (S/MAR) element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations which reduce their stability and can provoke cellular toxicity. Here, we present a new system, the Nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to their minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells by using Nano-S/MARs to stably restore the tumour suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that this class of DNA vector can be used to persistently modify a wide range of cells, providing sustained high levels of transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.
2021-12-12 | GSE142115 | GEO
Project description:Human induced pluripotent stem (iPS) cells have previously been derived from somatic cells using viral vectors that integrate transgenes into the genome. Genomic integration, however, can allow persistent leaky expression of the transgenes and can create insertional mutations, thus limiting the utility of these cells for both research and clinical applications. Here, we describe the derivation of human iPS cells free of vector and transgene sequences using non-integrating oriP/EBNA1-based episomal vectors. The resulting iPS cells are similar to human embryonic stem (ES) cells in both proliferative and developmental potential. These results demonstrate that reprogramming of human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors, and removes one important obstacle to the clinical applications of these cells. This SuperSeries is composed of the following subset Series:; GSE15175: Human induced pluripotent stem cells free of exogenous DNA are derived with episomal vectors (fig 1.c); GSE15176: Human induced pluripotent stem cells free of exogenous DNA are derived with episomal vectors (fig 4.a) Experiment Overall Design: Total 21 samples were analyzed to confirm the similarity of human iPS cells derived with episomal vectors with human ES cells, and a dissimilarity with fibroblasts. Experiment Overall Design: Refer to individual Series
2009-04-04 | E-GEOD-15148 | biostudies-arrayexpress
Project description:We examined genome-wide DNA methylation with 20 human iPSC lines derived from five different cell types,and 7 human ESC lines using Illumina’s Infinium HumanMethylation450K and compared among iPSCs by retrovirus, Sendai virus or episomal vectors.
2017-11-06 | GSE73938 | GEO
Project description:Two cell lines, BL41K3 and BJABK3, were chosen to perform a comprehensive screen for EBNA-2 target genes. Both cell lines are EBV negative B-cell lines and express a chimeric EBNA-2 protein fused to the hormone binding domain of the estrogen receptor (ER/EBNA-2). The cells are propagated in the absence of hormone 31,36. Estrogen stimulation activates ER/EBNA-2, which in turn activates well known EBNA-2 target genes like CD21. BL41K3 and BJABK3 were cultured in estrogen supplemented medium for 24h and gene expression profiles were established for unstimulated and stimulated cells. Experiment Overall Design: BJABK3: 3 biological replicates, for unstimulated and EBNA-2 induced cells, resp. Experiment Overall Design: BL41K3: 2 biological replicates, for unstimulated and EBNA-2 induced cell, resp.
2007-10-26 | E-GEOD-4525 | biostudies-arrayexpress
Project description:The genome of Epstein-Barr virus (EBV) encodes 86 proteins but only a limited set is expressed in EBV-growth transformed B cells, termed lymphoblastoid cell lines (LCLs). These cells proliferate via the concerted action of EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), some of which are rate limiting to establish a stable homeostasis of growth promoting and anti-apoptotic activities. We show here that EBV mutants, which lack the EBNA-3A gene, are impaired but can still initiate cell-cycle entry and proliferation of primary human B cells in contrast to an EBNA-2-deficient mutant virus. Surprisingly and in contrast to previous reports, these viral mutants are attenuated in growth transformation assays but give rise to permanently growing EBNA-3A negative B cell lines which exhibit reduced proliferation rates and elevated levels of apoptosis. Expression profiles of EBNA-3A deficient LCLs are characterized by 129 upregulated and 167 downregulated genes, which are significantly enriched for genes involved in apoptotic processes or cell cycle progression like the tumor suppressor gene p16/INK4A or might contribute to essential steps in the viral life cycle. In addition EBNA-3A cellular target genes remarkably overlap with previously identified targets of EBNA-2. Experiment Overall Design: Comparison of gene expression profiles from lymphoblastoid cell lines (LCLs) infected with EBNA-3A positive or negative EBV. LCLs were generated from three donors. A total of five EBNA-3A positive and nine negative LCLs were analysed.
2009-09-13 | E-GEOD-17908 | biostudies-arrayexpress