Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA389823 | ENA |
REPOSITORIES: ENA
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Project description:The advancement in translational research, such as gene therapy, stem cell engineering and molecular medicine, can be realized on the pretext of genetic engineering at cellular level. However, the challenge of inserting large transgene cassettes into the human genome could be an impediment to this advancement, which makes the cell engineering process a critical parameter. Current practices, including random integration transgenesis tools (viral and transposon-based) or specific genome manipulation tools (endonuclease-based) are suboptimal in delivering large transgenes and also pose safety concerns. To address this void, we had developed and reported a novel transgenesis tool derived from phage λ integrase that precisely recombines large plasmid DNA into an endogenous sequence found in about 1000 human Long INterspersed Elements-1 (LINE-1) in various human cell lines. As an improved extension to mitigate the biosafety concerns to the minimal and enhance the uptake and efficiency of transgene expression, we report here a technologically advanced λ-Int platform, wherein we show efficient derivation of seamless negatively supercoiled transgene vectors from the conventional plasmid DNA using in vitro intermolecular recombination followed by its integration into human LINE-1 elements via prototypical λ-Int system. Additionally, we have identified specific LINE-1 as preferred seamless vector insertion sites for λ-Int mediated transgenesis. Characteristically, this new platform achieves sustainable, high-level transgene expression as exemplified by a CD19 chimeric antigen receptor expression from targeted LINE-1 in hESCs cells. Therefore, we demonstrate that our novel seamless vector platform has the potential to be broadly applied across different applications in biotechnology and molecular medicine.
2017-06-10 | GSE99883 | GEO
Project description:Different attB or attP DNA libraries containing 7-bp random nucleotides were used for in vitro recombination mediated by the purified integrase from mv4 bacteriophage against their cognate wild-type attB or attP recombination site.
2023-12-10 | E-MTAB-13571 | biostudies-arrayexpress
Project description:The two obstacles that impede a wider application of genetically modified cells expressing therapeutic transgenes for ex vivo gene therapy are the immune mediated rejection of the transplanted cells, combined with their potential to cause iatrogenic oncogenesis. In this study we describe a new cellular vehicle for this form of therapy,; termed the cord lining epithelial cell (CLEC). CLECs are derived from the human amnion and incorporate many of the immunoregulatory functions associated with the fetal/maternal interface. We show that CLECs can be safely transfected by phage ÏC31 integrase to accomplish site-specific integration of a therapeutic human transgene. We also show that transplanted CLECs are not oncogenic in vivo and can be maintained in immunocompetent mice where acute xeno-rejection rapidly destroys other human cell types. Finally, we demonstrate the utility of CLECs for ex vivo gene therapy by delivering human coagulation factor 8 to mice with Hemophilia A. Experiment Overall Design: The transcriptome datasets of human umbilical cord lining epithelial cells were compared before(CLEC) and 1 month after(CLEC-GFP) phage integrase mediated integration of EGFP cDNA into the genome. Transcriptome datasets were generated in singles and genes differentiallty expressed in cells before and after phage integrase treatment were analysed. Genes differentially expressed by at least 2 fold as compared to untreated CLEC were considered to be significantly dysregulated. List of genes with significant dysregulation were used for further analysis and to used to determine if genomic integration events had resulted in any potential geno-toxicity.
2008-10-25 | E-GEOD-12510 | biostudies-arrayexpress
Project description:Co-IP of HIV-1 integrase proteins - WT, K258R and K258/264/266/273R. Integrase proteins were cloned into a mammalian expression vector (pJET). All proteins had an N-terminal HA-tag used for IP. As a negative control, cells were transfected with an empty HA vector. Co-IPs were done in HEK293T cells.
2022-05-20 | PXD031825 | Pride
Project description:We established a two-step approach to centromere-replacement (Figure 1A in outline, and Supplementary data in detail). FiC31 integrase was used to place a candidate sequence or an empty vector adjacent to the native centromere of chromosome 2 of CBS2777, and Bxb1 integrase was subsequently used to delete the native centromere.
2019-07-10 | GSE126511 | GEO
Project description:The two obstacles that impede a wider application of genetically modified cells expressing therapeutic transgenes for ex vivo gene therapy are the immune mediated rejection of the transplanted cells, combined with their potential to cause iatrogenic oncogenesis. In this study we describe a new cellular vehicle for this form of therapy, termed the cord lining epithelial cell (CLEC). CLECs are derived from the human amnion and incorporate many of the immunoregulatory functions associated with the fetal/maternal interface. We show that CLECs can be safely transfected by phage φC31 integrase to accomplish site-specific integration of a therapeutic human transgene. We also show that transplanted CLECs are not oncogenic in vivo and can be maintained in immunocompetent mice where acute xeno-rejection rapidly destroys other human cell types. Finally, we demonstrate the utility of CLECs for ex vivo gene therapy by delivering human coagulation factor 8 to mice with Hemophilia A. High-resolution copy number profiling was performed on genomic DNA of untreated (GSM315546 and GSM315713) and phage integrase modified CLECs (GSM315974 and GSM316895) using the Human Mapping 500K Array Set (Affymetrix) and the data analyzed using GeneChip Chromosome Copy Number Analysis Tool. Regions of copy number gain or loss were defined as having 3 consecutive SNPs concordant for significant copy number abnormalities. Log2 signal intensity ratios >0.3 and <-0.3 were criteria for significant copy number gain and loss, respectively.
2008-09-01 | E-GEOD-12629 | biostudies-arrayexpress
Project description:Investigation of whole genome gene expression level changes in HT1080 fibrosarcoma cell line after transfection CRABP1 gene and R131A CRABP1 mutant (arginine-alanine substitution in a protein active site, protein lacks the ability to interact with retinoic acid), compared to HT1080 line transfected with empty pLXSN vector.
2013-09-01 | GSE48673 | GEO
Project description:Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by the introduction of the transcription factors Oct4, Sox2, Klf4 and cMyc using various methods. Here, we describe a new approach for the derivation of murine iPSCs using a polycistronic non-viral inducible vector integrated into pseudo attP sites via the C31 integrase-mediated site-specific recombination and subsequent vector excision by Cre recombinase. The pluripotency of the derived iPSCs was proved by in vitro and in vivo tests. The derived transgene-free iPSCs reactivated the endogenous pluripotency genes like e.g. Oct4, Sox2 and Nanog and the global gene expression profiles of iPSCs lines are highly similar to ESCs and distinct from parental murine fibroblasts. We demonstrated the differentiation potential of iPSCs by generation cells of the three germ layers as well as we successfully created germline chimeric mice from transgene-free iPSCs. In this study, we presented an efficient method for the generation of transgene-free iPSCs using dual-recombinase technology. expression data of iPSCs/ESCs/MEFs
2014-03-29 | E-GEOD-51605 | biostudies-arrayexpress
Project description:Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by the introduction of the transcription factors Oct4, Sox2, Klf4 and cMyc using various methods. Here, we describe a new approach for the derivation of murine iPSCs using a polycistronic non-viral inducible vector integrated into pseudo attP sites via the C31 integrase-mediated site-specific recombination and subsequent vector excision by Cre recombinase. The pluripotency of the derived iPSCs was proved by in vitro and in vivo tests. The derived transgene-free iPSCs reactivated the endogenous pluripotency genes like e.g. Oct4, Sox2 and Nanog and the global gene expression profiles of iPSCs lines are highly similar to ESCs and distinct from parental murine fibroblasts. We demonstrated the differentiation potential of iPSCs by generation cells of the three germ layers as well as we successfully created germline chimeric mice from transgene-free iPSCs. In this study, we presented an efficient method for the generation of transgene-free iPSCs using dual-recombinase technology.
2014-03-29 | GSE51605 | GEO
Project description:The Gypsy-like element Ty3 inserts proximal to the transcription start sites of genes transcribed by RNA polymerase 3 (RNAP3). In this study, a random-barcode Ty3 was used to count Ty3 insertions at specific sites. Surprisingly, saturation transposition of the yeast genome showed that tDNAs even within isoacceptor families are targeted at widely different frequencies. Ectopic expression of Ty3 integrase showed that it localizes to integration targets independent of other Ty3 proteins. Binding of integrase, RNAP3 and factor Brf1 at individual targets did not differ to the same extent as integration. Metadata analysis showed that histone modification H3K4Ac correlated positively with insertion frequency. Targeting frequency could be reconstituted on high copy plasmids containing only 75 bp of 5’ flanking sequence plus the tDNA target. Weighting of insertions according to frequency identified an A/T-rich sequence followed by C as the site of gene-proximal strand transfer. This site lies immediately adjacent to the adenines of the RNAP3 transcription start site motif (CAA). Recent structures of DNA in RNAP3 initiation complexes show that in the initiation complex the transcription start site is sharply bent at the position adjacent to the gene-proximal Ty3 strand transfer. We propose that Ty3 integration occurs in two steps: in the first, host Brf1 engages integrase; in the second, integrase exploits YR flexibility, and that together, these steps determine the wide range of Ty3 targeting frequencies.
2018-04-15 | GSE97893 | GEO