Tet-on System Research Articles

Spatio-temporal control of targeted gene expression in combination with CRISPR/Cas and Tet-On systems in Medaka.

Spatial and temporal control of transgene expression is a powerful approach to understand gene functions in specific cells and tissues. The Tet-On system is a robust tool for controlling transgene expression spatially and temporally; however, few studies have examined whether this system can be applied to postembryonic stages of Medaka (Oryzias latipes) or other fishes. Here, we first improved a basal promoter sequence on the donor vector for a nonhomologous end joining (NHEJ)-based knock-in (KI) system. Next, using transgenic Medaka for establishing the Tet-On system by KI, we demonstrated that doxycycline administration for four or more days by feeding can be a stable and efficient method to achieve expression of the transduced reporter gene in adult fish. From these analyses, we propose an optimized approach for a spatio-temporal gene-expression system in the adult stage of Medaka and other small fishes.

T-ALL Cells as Tool Cells for CAR T Therapy.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic malignancy derived from T cells. Numerous CAR T therapies have been successfully applied to treat hematologic malignancies in the clinic. Nevertheless, there remain several challenges to the extensive application of CAR T cell therapy in T cell malignancies, especially in T-ALL. The main reason for CAR T therapy limitations is that T-ALL cells and normal T cells share antigens, which improves the difficulty of sorting pure T cells, resulting in product contamination, and would lead to CAR T cell fratricide. Thus, we considered creating a CAR on T-ALL tumor cells (CAR T-ALL) to prevent fratricide and eliminate tumor cells. We found that T-ALL cells transduced with CAR would actually commit fratricide. However, CAR T-ALL could kill only tumor cells on T-ALL cell lines, and other types of tumor cells had no killing function after being transferred with CAR. Furthermore, we created CD99 CAR with expression controlled by the Tet-On system on Jurkat cells, which could avoid the fratricide of CAR T-ALL during proliferation, ensuring the controllability of the killing time and effect. Jurkat transduced with a CAR-targeting antigen, which was expressed on other cancer cells, could kill other cancer cell lines, demonstrating that T-ALL cells could be used as tool cells for cancer therapy. Our study supplied a new feasible treatment regimen for cancer treatment in the clinic.

High TXNIP expression accelerates the migration and invasion of the GDM placenta trophoblast

IntroductionOur previous study has proofed the glucose sensitive gene-thioredoxin-interacting protein (TXNIP) expression was up in the placenta of the patients with gestational diabetes mellitus (GDM), but the pathological mechanisms underlying abnormal TXNIP expression in the placenta of patients with GDM is completely unclear and additional investigations are required to explain the findings we have observed. In the present study, we simulated the high TXNIP expression via introducing the Tet-On “switch” in vitro, approximate to its expression level in the real world, to explore the following consequence of the abnormal TXNIP.MethodsThe expression and localization of TXNIP in the placenta of GDM patients and the health control was investigated via immunofluorescent staining, western blot and RT-qPCR. Overexpression of TXNIP was achieved through transfecting Tet-on system to the human trophoblastic cell line-HTR-8/Svneo cell. TXNIP knockout was obtained via CRISPR-Cas9 method. The cell phenotype was observed via IncuCyte Imaging System and flow cytometry. The mechanism was explored via western blot and RT-qPCR.ResultsThe expression level of TXNIP in the GDM placenta was nearly 2–3 times higher than that in the control. The TXNIP located at trophoblastic cells of the placenta. When the expression of TXNIP was upregulated, the migration and invasion of the cells accelerated, but cell apoptosis and proliferation did not changed compared with the control group. Furthermore, the size of the TetTXNIP cells became larger, and the expression level of Vimentin and p-STAT3 increased in the TetTXNIP cells. All the changes mentioned above were opposite in the TXNIP-KO cells.ConclusionsAbnormal expression of TXNIP might be related to the impairment of the GDM placental function, affecting the migration and invasion of the placental trophoblast cells through STAT3 and Vimentin related pathway; thus, TXNIP might be the potential therapeutic target for repairing the placental dysfunction deficient in GDM patients.

Abstract 3727: Development of a new All-In-One inducible lentiviral shRNA/gRNA Vector

Abstract Tet-On is a powerful inducible system and a classical tool to regulate gene expression in mammalian cells. It has also been applied to regulate Pol III-driven transcription, such as shRNA or gRNA driven by a U6 or H1 promoter. However, all of the current versions of Tet-On shRNA vectors are based on H1-2O2 or U6-2O2 promoters, which are only compatible with first-generation tetracycline repressor TetR. In addition, these promoters have the problem of driving downstream transcription without the binding of the Tet regulatory protein. Here, we developed a new system that is built upon tetracycline activator protein, Tet-On 3G, combined with a new structure of Tet responsive promoter H1-4O4, which tightly regulates the downstream transcription of gRNA or shRNA. The responsiveness of our system to Doxycycline regulation is dramatically improved compared with the current versions. The new Tet-On system is further optimized into a compact structure to be compatible with the lentivirus package (All-In-One Lenti-Tet-On system), which still keeps the leaky expression at an undetectable level. Combined with all these features, the new generation of the Tet-On system offers broad applications in gene knocking down and genomic editing. Citation Format: Lipeng Wu, Hua Su, Justin Fellows, Mao Fu, Julian Heller, Brian Park, Dezhong Yin. Development of a new All-In-One inducible lentiviral shRNA/gRNA Vector. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3727.

Synthetic peptides for the precise transportation of proteins of interests to selectable subcellular areas.

Proteins, as gifts from nature, provide structure, sequence, and function templates for designing biomaterials. As first reported here, one group of proteins called reflectins and derived peptides were found to present distinct intracellular distribution preferences. Taking their conserved motifs and flexible linkers as Lego bricks, a series of reflectin-derivates were designed and expressed in cells. The selective intracellular localization property leaned on an RMs (canonical conserved reflectin motifs)-replication-determined manner, suggesting that these linkers and motifs were constructional fragments and ready-to-use building blocks for synthetic design and construction. A precise spatiotemporal application demo was constructed in the work by integrating RLNto2 (as one representative of a synthetic peptide derived from RfA1) into the Tet-on system to effectively transport cargo peptides into nuclei at selective time points. Further, the intracellular localization of RfA1 derivatives was spatiotemporally controllable with a CRY2/CIB1 system. At last, the functional homogeneities of either motifs or linkers were verified, which made them standardized building blocks for synthetic biology. In summary, the work provides a modularized, orthotropic, and well-characterized synthetic-peptide warehouse for precisely regulating the nucleocytoplasmic localization of proteins.

The maturation of iPS cell-derived brain microvascular endothelial cells by inducible-SOX18 expression

BackgroundBrain microvascular endothelial cells (BMECs) play a major role in the blood–brain barrier (BBB), and are critical for establishing an in vitro BBB model. Currently, iPSC-derived BMECs (iBMECs) have been used to construct in vitro BBB models with physiological barrier functions, such as high trans-endothelial electrical resistance (TEER) and expression of transporter proteins. However, the relatively low p-glycoprotein (P-gp) level and a decrease in the efflux ratio of its substrates in iBMECs suggest their immature nature. Therefore, more mature iBMECs by optimizing the differentiation induction protocol is beneficial for establishing a more reliable in vitro BBB model for studying central nervous system (CNS) drug transport.MethodsTo identify human brain endothelial cell fate-inducing factors, HUVEC was transfected with Zic3A-, Zic3B-, and Sox18-expressing lentivirus vector. Since SOX18 was found to induce BMEC properties, we used a Dox-inducible Tet-on system to express SOX18 during iBMEC differentiation and explored the impact of SOX18 expression on iBMEC maturation.ResultsSox18-mediated iBMECs achieved a higher TEER value than normal iBMECs (> 3000 Ω cm2). From day 6 to day 10 (d6–10 group), the iBMECs with SOX18 expression expressed a series of tight junction markers and showed upregulation of Mfsd2a, a specific marker of the BBB. The d6–10 group also expressed SLC2A1/Glut1 at levels as high as normal iBMECs, and upregulated ABCB1/P-gp and ABCC1/MRP1 expression. Moreover, Sox18-mediated iBMECs showed higher viability than normal iBMECs after puromycin treatment, indicating that SOX18 expression could upregulate P-gp activity in iBMECs.ConclusionsInducible SOX18 expression in iBMECs gained BBB phenotypes, including high TEER values and upregulation of tight junction-related genes, endothelial cell (EC) markers, BBB transporters, and higher cell viability after treatment with puromycin. Collectively, we provide a differentiation method for the maturation of human iPS cell-derived BMECs with SOX18 expression, describing its contribution to form an in vitro BBB model for CNS drug transport studies.

REGγ promotes mantle cell lymphoma cell apoptosis by downregulating NF-κB signaling.

Mantle cell lymphoma (MCL) is an aggressiveB-cell non-Hodgkin lymphoma (NHL). REGγ is important for tumor occurrence and development, but understanding of the specific role of REGγ in MCL is lacking. WeaimedtoidentifyREGγ effects on the proliferation and apoptosis of MCL cellsandclarifytheunderlying mechanisms. JEKO-1 cells stably transfected with a doxycycline-inducible Tet-On system expressed high levels of REGγ. JEKO-1 cells stably expressing shRNA-REGγ to reduce REGγ levels were constructed. Cell proliferation, apoptosis, and p-NF-κB, NF-κB, IkB, REGγ, p-STAT3, STAT3, and PSMB5 levels in transfected cells and in transfected cells treated with Stattic, that is a nonpeptidic small molecule exhibited to selectively inhibit signal transducer and activator of transcription factor 3 through blocking the function of its SH2 domain, were analyzed using western blotting. The proliferation of JEKO-1 cells was inhibited, and apoptosis was enhanced by increased expression of REGγ (P<0.01). REGγ inhibited MCL cell proliferation in a mouse tumor xenograft model by promoting apoptosis, increased the expression ofthe three IκB subunitsand inhibited NF-κB signaling. Overexpressed REGγ inhibited STAT3 and downregulated PSMB5 expression in MCL cells. Statticdownregulated PSMB5 and nuclear factor-kappa B (NF-κB) expressions and upregulated IκBε expression in JEKO-1 cells. We found that REGγ regulatedp-STAT3 expression by accelerating itshalf-life and downregulated the NF-κBsignaling pathway to promote MCL cell apoptosis bynegatively regulating STAT3-mediated PSMB5 expression andsubsequentlyupregulating IκB expression.

An optimized Tet-On system for conditional control of gene expression in sea urchins.

Sea urchins and other echinoderms are important experimental models for studying developmental processes. The lack of approaches for conditional gene perturbation, however, has made it challenging to investigate the late developmental functions of genes that have essential roles during early embryogenesis and genes that have diverse functions in multiple tissues. The doxycycline-controlled Tet-On system is a widely used molecular tool for temporally and spatially regulated transgene expression. Here, we optimized the Tet-On system to conditionally induce gene expression in sea urchin embryos. Using this approach, we explored the roles the MAPK signaling plays in skeletogenesis by expressing genes that perturb the pathway specifically in primary mesenchyme cells during later stages of development. We demonstrated the wide utility of the Tet-On system by applying it to a second sea urchin species and in cell types other than the primary mesenchyme cells. Our work provides a robust and flexible platform for the spatiotemporal regulation of gene expression in sea urchins, which will considerably enhance the utility of this prominent model system.

MYC Causes Multiple Myeloma Progression via Attenuating TP53-Induced MicroRNA-34 Expression.

MicroRNAs (miRNAs and miRs) are small (19-25 base pairs) non-coding RNAs with the ability to modulate gene expression. Previously, we showed that the miR-34 family is downregulated in multiple myeloma (MM) as the cancer progressed. In this study, we aimed to clarify the mechanism of miRNA dysregulation in MM. We focused particularly on the interaction between MYC and the TP53-miR34 axis because there is a discrepancy between increased TP53 and decreased miR-34 expressions in MM. Using the nutlin-3 or Tet-on systems, we caused wild-type (WT) p53 protein accumulation in human MM cell lines (HMCLs) and observed upregulated miR-34 expression. Next, we found that treatment with an Myc inhibitor alone did not affect miR-34 expression levels, but when it was coupled with p53 accumulation, miR-34 expression increased. In contrast, forced MYC activation by the MYC-ER system reduced nutlin-3-induced miR-34 expression. We also observed that TP53 and MYC were negatively correlated with mature miR-34 expressions in the plasma cells of patients with MM. Our results suggest that MYC participates in the suppression of p53-dependent miRNA expressions. Because miRNA expression suppresses tumors, its inhibition leads to MM development and malignant transformation.

Effect to Therapy of Sodium-Iodine Symporter Expression by Alpha-Ray Therapeutic Agent via Sodium/Iodine Symporter.

This study confirmed the effect of sodium/iodine symporter (NIS) expression on existing drugs by in vitro and in vivo tests using cultured cell lines. The tumor growth inhibitory effect of sodium astatide ([211At]NaAt) was evaluated by in vitro and in vivo tests using human thyroid cancer cells (K1, K1/NIS and K1/NIS-DOX). NIS expression in cancer cells was controlled using the Tet-On system. [131I]NaI was used as control existing drug. From the results of the in vitro studies, the mechanism of [211At]NaAt uptake into thyroid cancer cells is mediated by NIS, analogous to [131I]NaI, and the cellular uptake rate correlates with the expression level of NIS. [211At]NaAt's ability to inhibit colony formation was more than 10 times that of [131I]NaI per becquerel (Bq), and [211At]NaAt's DNA double-strand breaking (DSB) induction was more than ten times that of [131I]NaI per Bq, and [211At]NaAt was more than three times more cytotoxic than [131I]NaI (at 1000 kBq each). In vivo studies also showed that the tumor growth inhibitory effect of [211At]NaAt depended on NIS expression and was more than six times that of [131I]NaI per Bq.

Generation of a doxycycline-inducible ETV2 expression cell line using PiggyBac transposase system

E26 transformation-specific variant 2 (ETV2), as a member of Ets family of transcription factors, plays an important role in embryonic vasculogenesis, angiogenesis and hematopoiesis. Here, based on H9 cell line and PiggyBac technology, we generated a doxycycline-inducible ETV2 (Dox-ETV2-H9) embryonic stem cell (ESC) line. The Dox-ETV2-H9 cells maintain normal morphology, karyotype as well as high expression of pluripotent markers. In comparison with previous studies that employing costly ETV2 modRNA to improve differentiation from ESCs into endothelial cells (ECs), the tet-on system in Dox-ETV2-H9 cells could precisely manipulate ETV2 expression, which enables ECs differentiation process more efficient and controllable upon addition of doxycycline.

Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species

Conditional promoters allowing both induction and silencing of gene expression are indispensable for basic and applied research. The xylP promoter (pxylP) from Penicillium chrysogenum was demonstrated to function in various mold species including Aspergillus fumigatus. pxylP allows high induction by xylan or its degradation product xylose with low basal activity in the absence of an inducer. Here we structurally characterized and engineered pxylP in A. fumigatus to optimize its application. Mutational analysis demonstrated the importance of the putative TATA-box and a pyrimidine-rich region in the core promoter, both copies of a largely duplicated 91-bp sequence (91bpDS), as well as putative binding sites for the transcription factor XlnR and a GATA motif within the 91bpDS. In agreement, pxylP activity was found to depend on XlnR, while glucose repression appeared to be indirect. Truncation of the originally used 1643-bp promoter fragment to 725 bp largely preserved the promoter activity and the regulatory pattern. Integration of a third 91bpDS significantly increased promoter activity particularly under low inducer concentrations. Truncation of pxylP to 199 bp demonstrated that the upstream region including the 91bpDSs mediates not only inducer-dependent activation but also repression in the absence of inducer. Remarkably, the 1579-bp pxylP was found to act bi-bidirectionally with a similar regulatory pattern by driving expression of the upstream-located arabinofuranosidase gene. The latter opens the possibility of dual bidirectional use of pxylP. Comparison with a doxycycline-inducible TetOn system revealed a significantly higher dynamic range of pxylP. Taken together, this study identified functional elements of pxylP and opened new methodological opportunities for its application.

Modular Inducible Multigene Expression System for Filamentous Fungi.

Inducible promoters are indispensable elements when considering the possibility to modulate gene expression on demand. Desirable traits of conditional expression systems include their capacity for tight downregulation, high overexpression, and in some instances for fine-tuning, to achieve a desired product's stoichiometry. Although the number of inducible systems is slowly increasing, suitable promoters comprising these features are rare. To date, the concomitant use of multiple regulatable promoter platforms for controlled multigene expression has been poorly explored. This work provides pioneer work in the human pathogenic fungus Aspergillus fumigatus, wherein we investigated different inducible systems, elucidated three candidate promoters, and proved for the first time that up to three systems can be used simultaneously without interfering with each other. Proof of concept was obtained by conditionally expressing three antifungal drug targets within the ergosterol biosynthetic pathway under the control of the xylose-inducible PxylP system, the tetracycline-dependent Tet-On system, and the thiamine-repressible PthiA system. IMPORTANCE In recent years, inducible promoters have gained increasing interest for industrial or laboratory use and have become key instruments for protein expression, synthetic biology, and metabolic engineering. Constitutive, high-expressing promoters can be used to achieve high expression yields; however, the continuous overexpression of specific proteins can lead to an unpredictable metabolic burden. To prevent undesirable effects on the expression host's metabolism, the utilization of tunable systems that allow expression of a gene product on demand is indispensable. Here, we elucidated several excellent tunable promoter systems and verified that each can be independently induced in a single strain to ultimately develop a unique conditional multigene expression system. This highly efficient, modular toolbox has the potential to significantly advance applications in fundamental as well as applied research in which regulatable expression of several genes is a key requirement.

The Sall2 transcription factor promotes cell migration regulating focal adhesion turnover and integrin β1 expression.

SALL2/Sall2 is a transcription factor associated with development, neuronal differentiation, and cancer. Interestingly, SALL2/Sall2 deficiency leads to failure of the optic fissure closure and neurite outgrowth, suggesting a positive role for SALL2/Sall2 in cell migration. However, in some cancer cells, SALL2 deficiency is associated with increased cell migration. To further investigate the role of Sall2 in the cell migration process, we used immortalized Sall2 knockout (Sall2 -/- ) and Sall2 wild-type (Sall2 +/+ ) mouse embryonic fibroblasts (iMEFs). Our results indicated that Sall2 positively regulates cell migration, promoting cell detachment and focal adhesions turnover. Sall2 deficiency decreased cell motility and altered focal adhesion dynamics. Accordingly, restoring Sall2 expression in the Sall2 -/- iMEFs by using a doxycycline-inducible Tet-On system recovered cell migratory capabilities and focal adhesion dynamics. In addition, Sall2 promoted the autophosphorylation of Focal Adhesion Kinase (FAK) at Y397 and increased integrin β1 mRNA and its protein expression at the cell surface. We demonstrated that SALL2 increases ITGB1 promoter activity and binds to conserved SALL2-binding sites at the proximal region of the ITGB1 promoter, validated by ChIP experiments. Furthermore, the overexpression of integrin β1 or its blockade generates a cell migration phenotype similar to that of Sall2 +/+ or Sall2 -/- cells, respectively. Altogether, our data showed that Sall2 promotes cell migration by modulating focal adhesion dynamics, and this phenotype is associated with SALL2/Sall2-transcriptional regulation of integrin β1 expression and FAK autophosphorylation. Since deregulation of cell migration promotes congenital abnormalities, tumor formation, and spread to other tissues, our findings suggest that the SALL2/Sall2-integrin β1 axis could be relevant for those processes.

In Vitro Transfection of Up-Regulated Genes Identified in Favorable-Outcome Neuroblastoma into Cell Lines.

We previously used microarrays to show that high expression of DHRS3, NROB1, and CYP26A1 predicts favorable NB outcomes. Here, we investigated whether expression of these genes was associated with suppression of NB cell (SK-N-SH, NB12, and TGW) growth. We assessed morphology and performed growth, colony-formation, and migration assays, as well as RNA sequencing. The effects of the transient expression of these genes were also assessed with a tetracycline-controlled expression (Tet-On) system. Gene overexpression reduced cell growth and induced morphological senescence. Gene-expression analysis identified pathways involving cellular senescence and cell adhesion. In these cells, transduced gene dropout occurred during passage, making long-term stable gene transfer difficult. Tet-On-induced gene expression caused more pronounced cell-morphology changes. Specifically, DHRS3 and NROB1 led to rapid inhibition and arrest of cell growth, though CYP26A1 did not affect cell-growth rate or cell cycle. DHRS3 arrested the cell cycle by interacting with the all-trans-retinol pathway and drove differentiation and senescence in tumors. Overexpression of these genes reduced the malignant grade of these cells. A new therapeutic strategy might be the induction of these genes, as they suppress the growth of high-risk neuroblastoma and lead to differentiation and senescence.

Distepharinamide, a novel dimeric proaporphine alkaloid from Diploclisia glaucescens, inhibits the differentiation and proliferative expansion of CD4+Foxp3+ regulatory T cells

BackgroundCD4+Foxp3+ regulatory T cells (Tregs) represent the primary cellular mechanism of tumor immune evasion. Elimination of Treg activity by the pharmacological agent may enhance anti‐tumor immune responses. However, Treg-eliminating agents, especially those with small molecules, are rarely reported. PurposeTo identify small molecule inhibitors of Treg cells from natural products. MethodsCompounds from Diploclisia glaucescens were isolated by column chromatography, and structures were identified by spectroscopic evidence and quantum calculations. The tet-On system for Foxp3-GFP expression in Jurkat T cells was generated to screen Treg inhibitors based on Foxp3 expression. The effect of the compound on TNF-induced proliferative expansion of naturally occurring Tregs (nTregs) and TGF-β-induced generation of Tregs (iTregs) from naive CD4+ Tcells was further examined. ResultsA novel dimeric proaporphine alkaloid, designated as distepharinamide (DSA) with a symmetric structure isolated from the stems of D. glaucescens, restrained the doxycycline (Doxy)-induced Foxp3-tGFP expression, decreased the half-life of Foxp3 mRNA as well as reduced the mRNA levels of chemokine receptors (CCR4, CCR8 and CCR10) in Jurkat T cells with inducible Foxp3-tGFP expression. In lymphocytes or purified Tregs from wild-type C57BL/6 mice or from C57BL/6-Tg(Foxp3-DTR/EGFP)23.2Spar/Mmjax mice, DSA markedly inhibited TNF-induced proliferative expansion of Tregs present in the unfractionated CD4+ T cells, accompanied by the down-regulation of TNFR2, CD25 and CTLA4 expression on Tregs. Furthermore, DSA potently inhibited TGF-β-induced differentiation of Foxp3-expressing iTregs. Importantly, the expression of Foxp3 mRNA by both nTregs and iTregs was decreased by DSA treatment. Nevertheless, DSA at the same concentrations did not inhibit the proliferation of conventional CD4+ and CD8+ T cells stimulated by anti-CD3/CD28 antibodies. ConclusionDSA, a novel dimeric proaporphine alkaloid, potently inhibited the expansion of nTregs and generation of iTregs. Therefore, DSA or its analogs may merit further investigation as novel immunotherapeutic agents.

High expression of HIF-1α alleviates benzene-induced hematopoietic toxicity and immunosuppression in mice

Benzene exposure can cause pancytopenia and immunosuppression, leading to serious diseases such as aplastic anemia (AA) or acute myeloid leukemia (AML), but the underlying mechanism has not been fully elucidated. Hypoxia-inducible factor 1 (HIF-1) is an important transcription factor that regulates many downstream target genes. In this study, we reported a novel mechanism by which high expression of HIF-1α alleviated benzene toxicity. Mice with high expression of HIF-1α (HIF-1α+) were obtained by the Tet-on system and doxycycline induction, and they and wild-type (WT) mice were exposed to 150 mg/kg benzene for 0, 1, 3, 7, 10, 14, and 28 days. Dynamic changes in hematopoietic and immune-related indicators and the role of HIF-1α were explored. The level of white blood cells in mice reached the highest level on the third day, and immunity was activated and then suppressed within 10 days. Significant pancytopenia and immunosuppression occurred at 14 days and were more pronounced at 28 days. The levels of HIF-1α, EPO, VEGF, RORγt, and IL-17 in WT mice gradually decreased with increasing benzene exposure days, while the levels of Foxp3 and IL-10 increased. These changes were alleviated in HIF-1α+ mice. High expression of HIF-1α increased the levels of EPO and VEGF, which helped to maintain the stability of the hematopoietic microenvironment. Simultaneously, it attenuated benzene-induced immunosuppression by alleviating the Th17/Treg imbalance. HIF-1α is expected to be a new target for benzene-induced diseases such as AA and AML.

Inducible motor neuron differentiation of human induced pluripotent stem cells in vivo.

Transplantation of neural progenitor cells (NPCs) derived from human-induced pluripotent stem cells (hiPSCs) is one of the promising treatment strategies for motor neuron diseases (MNDs). However, the inefficiency in committed differentiation of NPCs in vivo limits its application. Here, we tried to establish a potential therapeutic strategy for MNDs by in vivo directional differentiation of hiPSCs engineered with motor neuron (MN) specific transcription factors and Tet-On system. We engineered hiPSCs with three MN-specific transcription factors and Tet-On system. The engineered cells were directly transplanted into immunodeficient mice through subcutaneous, intra-spinal cord and intracerebroventricular injections. Following doxycycline (Dox) induction, teratoma formation, and motor MN differentiation were evaluated. We generated genetically engineered hiPSCs, in which the expression of Ngn2, Isl1, and Lhx3 was controlled by a drug-inducible transgenic system. These cells showed normal pluripotency and proliferative capacity, and were able to directionally differentiate into mature motor neurons (MNs) and NPCs with high efficiency in spinal cords and cerebral lateral ventricles under the induction of Dox. The grafts showed long-term survival in the recipient mice without formation of teratoma. The induced mature MNs and NPCs were expected to replace the damaged endogenous MNs directly, and play a role of de novo stem cell stock for long-term neuron damage repair, respectively. Therefore, in vivo directional differentiation of the hiPSCs engineered with MN-specific transcription factors and Tet-On system via Dox induction could be a potential therapeutic strategy for MNDs with high efficacy and safety.

Limiting Transactivator Amounts Contribute to Transgene Mosaicism in Tet-On All-in-One Systems.

MicroRNAs play an essential role in cell homeostasis and have been proposed as therapeutic agents. One strategy to deliver microRNAs is to genetically engineer target cells to express microRNAs of interest. However, to control dosage and timing, as well as to limit potential side-effects, microRNAs' expression should ideally be under exogenous, inducible control. Conditional expression of miRNA-based short hairpin RNAs (shRNAmirs) via gene regulatory circuits such as the Tet-system is therefore a promising strategy to control shRNAmirs' expression in research and therapy. Single vector approaches like Tet-On all-in-one designs are more compatible with potential clinical applications by providing the Tet-On system components in a single round of genetic engineering. However, all-in-one systems often come at the expense of heterogeneous and unstable expression. In this study, we aimed to understand the causes that lead to such erratic transgene expression. By using a reporter cell, we found that the degree of heterogeneity mostly correlated with reverse tetracycline transactivator (rtTA) expression levels. Moreover, the targeted integration of a potent rtTA expression cassette into a genomic safe harbor locus functionally rescued previously silenced rtTA-responsive transcription units. Overall, our results suggest that ensuring homogenous and stable rtTA expression is essential for the robust and reliable performance of future Tet-On all-in-one designs.

Transgenically expressed Helicobacter pylori CagA in vascular endothelial cells accelerates arteriosclerosis in mice

Arteriosclerosis is intimately associated with cardiovascular diseases. Recently, evidence accumulated that infection with Helicobacter pylori cagA-positive strains, which causes gastritis, peptic ulceration, and gastric cancer, is also involved in the development of arteriosclerosis. The cagA-encoded CagA protein is injected into the attached gastric epithelial cells via the type IV secretion system. We previously showed that CagA-containing exosomes are secreted from CagA-injected gastric epithelial cells and enter the systemic blood circulation, delivering CagA into endothelial cells. In the present study, transgenic mice were established in which CagA was selectively expressed in endothelial cells by Cre-loxP system. Treatment of the mice with a high-fat diet revealed that atherogenic lesions were induced in mice expressing CagA in vascular endothelial cells but not in CagA-nonexpressing mice. To investigate the effects of CagA on endothelial cells, we also established conditional CagA-expressing human vascular endothelial cells using the Tet-on system. Upon induction of CagA, a dramatic change in cell morphology was observed that was concomitantly associated with the loss of the endothelial cells to form tube-like structures. Induction of CagA also activated the pro-inflammatory transcription factor STAT3. Thus, exosome-delivered CagA deregulates signals that activates STAT3 in endothelial cells, which accelerates inflammation that promotes arteriosclerosis/atherosclerosis.