An improved Tet-on system in microRNA overexpression and CRISPR/Cas9-mediated gene editing

Kang, Kang; Huang, Lian; Li, Qing; Liao, Xiaoyun; Dang, Quanjin; Yang, Yi; Luo, Jun; Zeng, Yan; Li, Li; Gou, Deming

doi:10.1186/s40104-019-0354-5

Cited by 18 publications

(19 citation statements)

References 37 publications

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“…2 A,B). It has previously been reported that expression of rtTA-M2 by strong promoters compromises inducible expression 16 , 17 . We therefore relocated the original rtTA-M2 sequence distal to the RPBSA and downstream from either one (pSBtet-2) or two (pSBtet-3) additional GOI.…”

Section: Resultsmentioning

confidence: 99%

Optimisation of Tet-On inducible systems for Sleeping Beauty-based chimeric antigen receptor (CAR) applications

Rad

Poudel

Tan

et al. 2020

Sci Rep

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Regulated expression of genetic elements that either encode polypeptides or various types of functional RNA is a fundamental goal for gene therapy. Inducible expression may be preferred over constitutive promoters to allow clinician-based control of gene expression. Existing Tet-On systems represent one of the tightest rheostats for control of gene expression in mammals. However, basal expression in absence of tetracycline compromises the widespread application of Tet-controlled systems in gene therapy. We demonstrate that the order of P2A-linked genes of interest was critical for maximal response and tightness of a chimeric antigen receptor (CAR)-based construct. The introduction of G72V mutation in the activation region of the TetR component of the rtTA further improved the fold response. Although the G72V mutation resulted in a removal of a cryptic splice site within rtTA, additional removal of this splice site led to only a modest improvement in the fold-response. Selective removal of key promoter elements (namely the BRE, TATA box, DPE and the four predicted Inr) confirmed the suitability of the minimal CMV promoter and its downstream sequences for supporting inducible expression. The results demonstrate marked improvement of the rtTA based Tet-On system in Sleeping Beauty for applications such as CAR T cell therapy.

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Section: Resultsmentioning

confidence: 99%

Optimisation of Tet-On inducible systems for Sleeping Beauty-based chimeric antigen receptor (CAR) applications

Rad

Poudel

Tan

et al. 2020

Sci Rep

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“…In recent years, much attention has been dedicated to explore the diagnostic significance of miRNAs in cancer . In 2002, Calin et al drew a relationship between cancer‐ and tissue‐derived miRNA where it was demonstrated that miRNA cluster miR 15/16 was downexpressed in chronic lymphocytic leukemia.…”

Section: Diagnostic and Prognostic Roles Of Rnamentioning

confidence: 99%

“…RT‐qPCR is a sensitive and widely used method for RNA analysis due to the advantages associated with the requirement of relatively less starting RNA, wide dynamic range, and better accuracy . Over the past several decades, a great number of RT‐qPCR‐based RNA expression analysis methods have been developed and also commonly used in clinical trials . In 2005, Chen et al developed one of the well‐known methods for miRNA analysis, referred to as TaqMan miRNA assay ( Figure I).…”

Section: Rna Biomarker Detection Technologiesmentioning

confidence: 99%

RNA Biomarkers: Diagnostic and Prognostic Potentials and Recent Developments of Electrochemical Biosensors

et al. 2017

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Ribonucleic acids (RNAs) are considered as effective and minimally invasive biomarkers for disease diagnosis and prognosis due to their critical role in the regulation of different cellular processes. Over the past several years, the rapid progress in RNA biomarker research has resulted in the development of a large number of high‐performance RNA‐detection methods. Most of these methods are based on molecular‐biology techniques such as quantitative reverse transcription polymerase chain reaction (RT‐qPCR), microarrays, and RNA sequencing. In recent years, considerable attention has also been dedicated to developing RNA biosensors, exploiting micro‐ and nanofabrication technologies, and various readout strategies, including electrochemical and optical transducers. Here, the recent developments of RNA biosensors are concisely reviewed with a special emphasis on electrochemical‐detection approaches. The following points are also highlighted: i) all the types of clinically relevant RNAs (mRNAs, miRNAs, lncRNAs) and their diagnostic and prognostic potential in cancer are outlined, ii) major challenges associated with current techniques are identified, followed by a critical analysis of how these challenges have been addressed by different biosensing approaches, and iii) the current requirements that still need to be met for effective screening of RNA biomarkers in both research and clinical settings.

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“…Here we focus on characterizing the DOX response in the renal epithelial cell line, mIMCD3. The cells have been engineered to include a tetracycline-inducible gene expression system (Schlimpert et al, 2018;Lashhab et al, 2019;Kang et al, 2019), and the model has proven popular for studying genes in a renal epithelial environment. Here we examine how DOX affects the transcriptome profiles of mIMCD3 cells with RNA-Seq.…”

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confidence: 99%

Doxycycline Changes the Transcriptome Profile of mIMCD3 Renal Epithelial Cells

et al. 2021

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Tetracycline-inducible gene expression systems have been used successfully to study gene function in vivo and in vitro renal epithelial models but the effects of the common inducing agent, doxycycline (DOX), on gene expression are not well appreciated. Here, we evaluated the DOX effects on the transcriptome of a widely used renal epithelial cell model, mIMCD3 cells, to establish a reference. Cells were grown on permeable filter supports in the absence and presence of DOX (3 or 6 days), and genome-wide transcriptome profiles were assessed using RNA-Seq. We found DOX significantly altered the transcriptome profile, changing the abundance of 1,549 transcripts at 3 days and 2,643 transcripts at 6 days. Within 3 days of treatment, DOX significantly decreased the expression of multiple signaling pathways (ERK, cAMP, and Notch) that are associated with cell proliferation and differentiation. Genes associated with cell cycle progression were subsequently downregulated in cells treated with DOX for 6 days, as were genes involved in cellular immune response processes and several cytokines and chemokines, correlating with a remarkable repression of genes encoding cell proliferation markers. The results provide new insight into responses of renal epithelial cells to DOX and a establish a resource for DOX-mediated gene expression systems.

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An improved Tet-on system in microRNA overexpression and CRISPR/Cas9-mediated gene editing

Cited by 18 publications

References 37 publications

Optimisation of Tet-On inducible systems for Sleeping Beauty-based chimeric antigen receptor (CAR) applications

Optimisation of Tet-On inducible systems for Sleeping Beauty-based chimeric antigen receptor (CAR) applications

RNA Biomarkers: Diagnostic and Prognostic Potentials and Recent Developments of Electrochemical Biosensors

Doxycycline Changes the Transcriptome Profile of mIMCD3 Renal Epithelial Cells

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