Reverse Transactivator Research Articles

Conditional KCa3.1-transgene induction in murine skin produces pruritic eczematous dermatitis with severe epidermal hyperplasia and hyperkeratosis.

Ion channels have recently attracted attention as potential mediators of skin disease. Here, we explored the consequences of genetically encoded induction of the cell volume-regulating Ca2+-activated KCa3.1 channel (Kcnn4) for murine epidermal homeostasis. Doxycycline-treated mice harboring the KCa3.1+-transgene under the control of the reverse tetracycline-sensitive transactivator (rtTA) showed 800-fold channel overexpression above basal levels in the skin and solid KCa3.1-currents in keratinocytes. This overexpression resulted in epidermal spongiosis, progressive epidermal hyperplasia and hyperkeratosis, itch and ulcers. The condition was accompanied by production of the pro-proliferative and pro-inflammatory cytokines, IL-β1 (60-fold), IL-6 (33-fold), and TNFα (26-fold) in the skin. Treatment of mice with the KCa3.1-selective blocker, Senicapoc, significantly suppressed spongiosis and hyperplasia, as well as induction of IL-β1 (-88%) and IL-6 (-90%). In conclusion, KCa3.1-induction in the epidermis caused expression of pro-proliferative cytokines leading to spongiosis, hyperplasia and hyperkeratosis. This skin condition resembles pathological features of eczematous dermatitis and identifies KCa3.1 as a regulator of epidermal homeostasis and spongiosis, and as a potential therapeutic target.

Conditional Immortalization of Lymphoid Progenitors via Tetracycline-Regulated LMO2 Expression.

Conditional immortalization of hematopoietic progenitors through lentiviral expression of selected transcription factors in hematopoietic stem and progenitor cells provides a promising tool to study stem cell and leukemia biology. In this study, to generate conditionally immortalized lymphoid progenitor (ciLP) cell lines, murine hematopoietic progenitor cells were transduced with an inducible lentiviral "all-in-one" vector expressing LMO2 under doxycycline (DOX) stimulation and the reverse tetracycline-regulated transactivator (rtTA3). For selection of LMO2-expressing ciLPs (LMO2-ciLPs) and longitudinal manipulation in T cell differentiation lymphoid conditions, we developed a robust approach based on coculture with OP9-DL1 stromal cells and improved cytokine conditions allowing a controlled balance between cell proliferation and differentiation in vitro. LMO2-ciLP cell lines with the highest proliferation, vector copy number, and similar insertion pattern were selected for LMO2 "on/off" in vitro study. LMO2 expression under DOX induction resulted in a double negative (DN) 2 differentiation arrest and a propagation of CD44+CD25- myeloid cell population characterized by lymphoid and myeloid phenotypes, respectively. Both DN2 and CD44+CD25- myeloid cell subpopulations expressed c-KIT, suggesting that LMO2-ciLPs were similar to uncommitted progenitors under DOX supplementation. DOX removal resulted in cessation of ectopic LMO2 expression and LMO2-ciLPs continued T cell lymphoid differentiation accompanied by c-KIT downregulation and interleukin 7 receptor expression. Switching off LMO2 expression was accompanied by increased Notch signaling and significant reduction of the CD44+CD25- myeloid cell population under T cell differentiation lymphoid conditions. Although vector insertions in cooperation with LMO2 expression could influence the fate of LMO2-ciLPs and additional experiments are required to evaluate it, our approach provides a promising tool to investigate mechanisms underlying stem cell, leukemia, and lymphocyte biology, leading to novel approaches for disease modeling and therapy evaluation.

Abstract PR16: Normal and neoplastic tissues with partial Hus1 impairment show hypersensitivity to cisplatin in vivo

Abstract The DNA damage response (DDR) factor HUS1 is vital for proper functioning of the ATR checkpoint pathway. Following DNA damage, HUS1 forms a heterotrimeric complex with RAD9 and RAD1 and works with other checkpoint and scaffold proteins to activate ATR and CHK1 kinases. HUS1 also has separate functions that promote DNA repair. Due to its essential nature and diverse roles in genome maintenance, we hypothesized that HUS1 would be critical in the response of normal and neoplastic tissues to the DNA-damaging chemotherapeutic, cisplatin. Cisplatin is a widely used chemotherapeutic that damages DNA via platinated adducts, which form intrastrand and interstrand crosslinks. To test our hypothesis, we made use of a mouse model whereby a hypomorphic Hus1 allele is combined with a null allele in order to achieve partial impairment (Hus1neo/Δ1n) in vivo. Hus1+ and Hus1neo/Δ1n FVB mice were administered high dose cisplatin intraperitoneally (13.5 mg/kg). Body weights were recorded daily and mice were euthanized when humane intervention criteria were met. Hus1neo/Δ1n mice had reduced 7-day survival (P = .0071) and increased weight loss (P = .0485) when compared to Hus1+ mice. In order to determine if neoplastic tissues had a similar requirement for HUS1 in response to DNA damage inflicted by cisplatin, we combined Hus1 impairment with K-ras oncogene activation in vivo. K-RAS is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC). Mutations in this oncogene, the majority of which occur in codon 12, affect 25–30% of western NSCLC patients. K-RAS encodes for a membrane-bound GTPase that regulates cell growth, differentiation, and survival. In our model, activation of oncogenic K-ras is restricted to the lung and is achieved via a tetracycline-controlled reverse transactivator linked to the CCSP promoter. In the presence of doxycycline, mutant Kras4bG12D is expressed in type II alveolar epithelial cells, and lung tumors develop in a regulatable, predictable manner. Mice with wild-type or partial Hus1 impairment and simultaneous oncogenic K-ras activation were screened for lung tumor development via computed tomography (micro-CT), then treated with maximum-tolerated dose (MTD) cisplatin, and assessed for response according to semiautomated volumetric methods and the clinical standard, RECIST (Response Evaluation Criteria in Solid Tumors). Using either histologic evaluation or micro-CT imaging, mice with partial Hus1 impairment have a decreased tumor burden at 1- and 3- weeks after cisplatin treatment, as compared to mice with wild type Hus1. Overall, this data identifies a role for HUS1 in the response of normal and neoplastic tissues to the DNA-damaging chemotherapeutic, cisplatin. Understanding tissue specific responses to cisplatin is important for determining how therapeutics targeting the DDR will work cooperatively with traditional cytotoxic agents in the treatment of cancer. This abstract is also being presented as Poster B08. Citation Format: Terese E. Noe, Cleo C. Siderides, Aitor Gallastegui, James M. Cheung, Tina Abratte, Kelly R. Hume. Normal and neoplastic tissues with partial Hus1 impairment show hypersensitivity to cisplatin in vivo [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR16.

Over-expression of Slc30a8/ZnT8 selectively in the mouse α cell impairs glucagon release and responses to hypoglycemia.

BackgroundThe human SLC30A8 gene encodes the secretory granule-localised zinc transporter ZnT8 whose expression is chiefly restricted to the endocrine pancreas. Single nucleotide polymorphisms (SNPs) in the human SLC30A8 gene have been associated, through genome-wide studies, with altered type 2 diabetes risk. In addition to a role in the control of insulin release, recent studies involving targeted gene ablation from the pancreatic α cell (Solomou et al., J Biol Chem 290(35):21432-42) have also implicated ZnT8 in the control of glucagon release. Up to now, however, the possibility that increased levels of the transporter in these cells may impact glucagon secretion has not been explored.MethodsHere, we use a recently-developed reverse tetracyline transactivator promoter-regulated ZnT8 transgene to drive the over-expression of human ZnT8 selectively in the α cell in adult mice. Glucose homeostasis and glucagon secretion were subsequently assessed both in vivo during hypoglycemic clamps and from isolated islets in vitro.ResultsDoxyclin-dependent human ZnT8 mRNA expression was apparent in both isolated islets and in fluorescence-activated cell sorting- (FACS) purified α cells. Examined at 12 weeks of age, intraperitoneal glucose (1 g/kg) tolerance was unchanged in transgenic mice versus wild-type littermates (n = 8-10 mice/genotype, p > 0.05) and sensitivity to intraperitoneal insulin (0.75U/kg) was similarly unaltered in transgenic animals. In contrast, under hyperinsulinemic-hypoglycemic clamp, a ~45 % (p < 0.001) reduction in glucose infusion rate was apparent, and glucagon release was significantly (~40 %, p < 0.01) impaired, in transgenic mice. Correspondingly, examined in vitro, glucagon secretion was significantly reduced (~30 %, p < 0.05) from transgenic versus control islets at low, stimulatory glucose concentrations (1 mM, p < 0.05) but not at high glucose (17 mM) glucose (p > 0.05). Over-expression of ZnT8 in glucagonoma-derived αTC1-9 cells increased granule free Zn2+ concentrations consistent with a role for Zn2+ in this compartment in the action of ZnT8 on glucagon secretion.ConclusionsIncreased ZnT8 expression, and a likely increase in intragranular free Zn2+ concentration, is deleterious in pancreatic α cells for stimulated glucagon release. These data provide further evidence that type 2 diabetes-associated polymorphisms in the SLC30A8/ZnT8 gene may act in part via alterations in glucagon release and suggest that ZnT8 activation may restrict glucagon release in some settings.

Conditional knockdown of BCL2A1 reveals rate-limiting roles in BCR-dependent B-cell survival.

Bcl2 family proteins control mitochondrial apoptosis and its members exert critical cell type and differentiation stage-specific functions, acting as barriers against autoimmunity or transformation. Anti-apoptotic Bcl2a1/Bfl1/A1 is frequently deregulated in different types of blood cancers in humans but its physiological role is poorly understood as quadruplication of the Bcl2a1 gene locus in mice hampers conventional gene targeting strategies. Transgenic overexpression of A1, deletion of the A1-a paralogue or constitutive knockdown in the hematopoietic compartment of mice by RNAi suggested rate-limiting roles in lymphocyte development, granulopoiesis and mast cell activation. Here we report on the consequences of conditional knockdown of A1 protein expression using a reverse transactivator (rtTA)-driven approach that highlights a critical role for this Bcl2 family member in the maintenance of mature B-cell homeostasis. Furthermore, we define the A1/Bim (Bcl-2 interacting mediator of cell death) axis as a target of key kinases mediating B-cell receptor (BCR)-dependent survival signals, such as, spleen tyrosine kinase (Syk) and Brutons tyrosine kinase (Btk). As such, A1 represents a putative target for the treatment of B-cell-related pathologies depending on hyperactivation of BCR-emanating survival signals and loss of A1 expression accounts, in part, for the pro-apoptotic effects of Syk- or Btk inhibitors that rely on the ‘BH3-only' protein Bim for cell killing.

An Efficient and Versatile System for Visualization and Genetic Modification of Dopaminergic Neurons in Transgenic Mice.

Background & AimsThe brain dopaminergic (DA) system is involved in fine tuning many behaviors and several human diseases are associated with pathological alterations of the DA system such as Parkinson’s disease (PD) and drug addiction. Because of its complex network integration, detailed analyses of physiological and pathophysiological conditions are only possible in a whole organism with a sophisticated tool box for visualization and functional modification.Methods & ResultsHere, we have generated transgenic mice expressing the tetracycline-regulated transactivator (tTA) or the reverse tetracycline-regulated transactivator (rtTA) under control of the tyrosine hydroxylase (TH) promoter, TH-tTA (tet-OFF) and TH-rtTA (tet-ON) mice, to visualize and genetically modify DA neurons. We show their tight regulation and efficient use to overexpress proteins under the control of tet-responsive elements or to delete genes of interest with tet-responsive Cre. In combination with mice encoding tet-responsive luciferase, we visualized the DA system in living mice progressively over time.ConclusionThese experiments establish TH-tTA and TH-rtTA mice as a powerful tool to generate and monitor mouse models for DA system diseases.

Tightly regulated 'all-in-one' lentiviral vectors for protection of human hematopoietic cells from anticancer chemotherapy.

Successful application of gene therapy strategies may require stringently regulated transgene expression. Along this line, we describe a doxycycline (Dox)-inducible 'all-in-one' lentiviral vector design using the pTET-T11 (TII) minimal-promoter and a reverse transactivator protein (rtTA2S-M2) driven by the phosphoglycerate kinase promoter allowing for tight regulation of transgene expression (Lv.TII vectors). Vector design was evaluated in human hematopoietic cells in the context of cytidine deaminase (hCDD)-based myeloprotective gene therapy. Upon Dox administration, a rapid (16-24 h) and dose-dependent (>0.04 μg ml(-1) Dox) onset of transgene expression was detected in Lv.TII.CDD gene-modified K562 cells as well as in primary human CD34(+) hematopoietic cells. Importantly, in both cell models low background transgene expression was observed in the absence of Dox. Functionality of Dox-inducible hCDD expression was demonstrated by >10-fold increase in cytosine arabinoside (1-β-d-arabinofuranosylcytosine, Ara-C) resistance of Lv.TII.CDD-transduced K562 cells. In addition, Lv.TII.CDD-transduced CD34(+)-derived myeloid cells were protected from up to 300 nm Ara-C (control affected from 50 nm onwards). These data clearly demonstrate the suitability of our self-inactivating lentiviral vector to induce robust, tightly regulated transgene expression in human hematopoietic cells with minimal background activity and highlight the potential of our construct in myeloprotective gene therapy strategies.

The PDZ Motif of the α1C Subunit Is Not Required for Surface Trafficking and Adrenergic Modulation of CaV1.2 Channel in the Heart

Voltage-gated Ca(2+) channels play a key role in initiating muscle excitation-contraction coupling, neurotransmitter release, gene expression, and hormone secretion. The association of CaV1.2 with a supramolecular complex impacts trafficking, localization, turnover, and, most importantly, multifaceted regulation of its function in the heart. Several studies hint at an important role for the C terminus of the α1C subunit as a hub for multidimensional regulation of CaV1.2 channel trafficking and function. Recent studies have demonstrated an important role for the four-residue PDZ binding motif at the C terminus of α1C in interacting with scaffold proteins containing PDZ domains, in the subcellular localization of CaV1.2 in neurons, and in the efficient signaling to cAMP-response element-binding protein in neurons. However, the role of the α1C PDZ ligand domain in the heart is not known. To determine whether the α1C PDZ motif is critical for CaV1.2 trafficking and function in cardiomyocytes, we generated transgenic mice with inducible expression of an N-terminal FLAG epitope-tagged dihydropyridine-resistant α1C with the PDZ motif deleted (ΔPDZ). These mice were crossed with α-myosin heavy chain reverse transcriptional transactivator transgenic mice, and the double-transgenic mice were fed doxycycline. The ΔPDZ channels expressed, trafficked to the membrane, and supported robust excitation-contraction coupling in the presence of nisoldipine, a dihydropyridine Ca(2+) channel blocker, providing functional evidence that they appropriately target to dyads. The ΔPDZ Ca(2+) channels were appropriately regulated by isoproterenol and forskolin. These data indicate that the α1C PDZ motif is not required for surface trafficking, localization to the dyad, or adrenergic stimulation of CaV1.2 in adult cardiomyocytes.

Low levels of the reverse transactivator fail to induce target transgene expression in vascular smooth muscle cells.

Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease with multiple features that are suggestive of premature aging. Most patients with HGPS carry a mutation on one of their copies of the LMNA gene. The LMNA gene encodes the lamin A and lamin C proteins, which are the major proteins of the nuclear lamina. The organs of the cardiovascular system are amongst those that are most severely affected in HGPS, undergoing a progressive depletion of vascular smooth muscle cells, and most children with HGPS die in their early teens from cardio-vascular disease and other complications from atherosclerosis. In this study, we developed a transgenic mouse model based on the tet-ON system to increase the understanding of the molecular mechanisms leading to the most lethal aspect of HGPS. To induce the expression of the most common HGPS mutation, LMNA c.1824C>T; p.G608G, in the vascular smooth muscle cells of the aortic arch and thoracic aorta, we used the previously described reverse tetracycline-controlled transactivator, sm22α-rtTA. However, the expression of the reverse sm22α-transactivator was barely detectable in the arteries, and this low level of expression was not sufficient to induce the expression of the target human lamin A minigene. The results from this study are important because they suggest caution during the use of previously functional transgenic animal models and emphasize the importance of assessing transgene expression over time.

A Novel Conditional Mouse Model For MLL-ENL Induced Acute Leukemia

The t(11;19) translocation leading to the MLL-ENL fusion is recurrently found in pediatric and adult de novo and therapy related mixed-lineage acute leukemia and is often associated with a poor prognosis. Previous studies have shown that (retroviral) overexpression of MLL-ENL potently immortalizes bone marrow cells in vitro and induces a lethal acute myeloid leukemia (AML) in mice. To establish a mouse model that phenocopies more closely the human disease, we generated conditional transgenic mice in which the expression of MLL-ENL is controlled by doxycycline (DOX) through a stably integrated reverse tet-responsive transactivator (rtTA). Induction of MLL-ENL expression in newborn or adult mice resulted in a leukemic phenotype that phenocopied pre-B- and myeloid mixed lineage leukemia as observed in most patients with MLL-ENL. The diseased mice displayed excessive splenomegaly, massive lymph node as well as multiple organ infiltration by two co-existing types of blasts mostly expressing higher or lower levels of B220 and Gr1/Mac1 and similar levels of c-kit. Expression of the fusion gene and disease induction was DOX dosage dependent and reversible upon DOX removal. Despite significantly lower fusion gene expression levels as we observed in retroviral systems the median latency for the development of the disease in this model (104.3±16.9 days) was comparable to them (62±10.4 days) and much shorter than any of the previously reported MLL-ENL knock-in mouse models (> 1 year). Continuous ex vivo expression of MLL-ENL provided bone marrow and fetal liver hematopoietic cells with a strong self-renewal capacity and caused the accumulation of immature blast-like cells upon serial replating in methylcellulose cultures. In the presence of factors favoring myelopoiesis, like IL-3, DOX removal resulted in a complete differentiation towards the granulocytic-monocytic, lineages expressing high levels of Mac1/Gr-1, whereas IL-7 favored differentiation towards the B-cell lineage characterized by the expression of high levels of B220. In addition, MLL-ENL induced a DOX dependent aberrant self renewal capacity and a differentiation block in methylcellulose cultures of hematopoietic stem cells (Lin- c-kit+ Sca-1+, LSK) and various progenitors including common lymphoid progenitor (CLP) and granulocyte-macrophage progenitor (GMP) cells. Interestingly, MLL-ENL expression preferentially expanded LSK- rather than GMP-derived cells as assayed by growth curves in long-term (> 1 month) liquid cultures in the presence of cytokines in vitro. In line with this observation, in vivo, expression of MLL-ENL in long-term hematopoietic stem cells (LT-HSC) induced an aggressive mixed lineage leukemia characterized by the presence of two distinguishable populations of blasts, whereas induction in GMPs never induced a disease. These data suggest that MLL-ENL preferentially transforms hematopoietic stem cells rather than more differentiated progenitors. Thus, this novel transgenic mouse model for MLL-ENL induced acute leukemia closely recapitulates the human disease and combines the advantages of the existing knock-in and retroviral models. This model allowed us to demonstrate that in contrast to the MLL-AF9 fusion, that preferentially immortalizes GMPs, MLL-ENL preferentially transforms HSCs. We anticipate that our model will be a valuable tool to study the cellular origin and to search for and/or validate novel therapeutic targets for MLL-ENL induced acute leukemia. Disclosures:No relevant conflicts of interest to declare.

Generation and characterization of an inducible transgenic model for studying mouse esophageal biology

BackgroundTo facilitate the in vivo study of esophageal (stem) cell biology in homeostasis and cancer, novel mouse models are necessary to elicit expression of candidate genes in a tissue-specific and inducible fashion. To this aim, we developed and studied a mouse model to allow labeling of esophageal cells with the histone 2B-GFP (H2B-GFP) fusion protein.ResultsFirst, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the promoter (ED-L2) of the Epstein-Barr virus (EBV) gene encoding the latent membrane protein-1 (LMP-1). The newly generated ED-L2-rtTA2-M2 (ED-L2-rtTA) mice were then bred with the previously developed tetO-HIST1H2BJ/GFP (tetO-H2B-GFP) model to assess inducibility and tissue-specificity. Expression of the H2B-GFP fusion protein was observed upon doxycycline induction but was restricted to the terminally differentiated cells above the basal cell layer. To achieve expression in the basal compartment of the esophagus, we subsequently employed a different transgenic model expressing the reverse transactivator rtTA2S-M2 under the control of the ubiquitous, methylation-free CpG island of the human hnRNPA2B1-CBX3 gene (hnRNP-rtTA). Upon doxycycline administration to the compound hnRNP-rtTA/tetO-H2B-GFP mice, near-complete labeling of all esophageal cells was achieved. Pulse-chase experiments confirmed that complete turnover of the esophageal epithelium in the adult mouse is achieved within 7–10 days.ConclusionsWe show that the esophagus-specific promoter ED-L2 is expressed only in the differentiated cells above the basal layer. Moreover, we confirmed that esophageal turn-over in the adult mouse does not exceed 7–10 days.

Depot formation of doxycycline impairs Tet-regulated gene expression in vivo

The tetracycline (Tet) system is widely used for regulation of gene expression in vitro and in vivo. We constructed C57BL/6 transgenic mice (rtTA-CM2) with strong and ubiquitous reverse transactivator (rtTA2(S)-M2) gene expression. rtTA-CM2 mice were crossed to Tet-responsive reporter mice (LC-1) conditionally expressing the firefly luciferase (FLuc) gene under control of a Tet-responsive element, which allowed sensitive quantification of the transactivator activity by bioluminescent imaging. Following doxycycline (dox) application, up to 10(5)-fold increase in BL signal was measured. rtTA activity was inducible in most analyzed organs. After dox withdrawal the BL signal decreased significantly but did not disappear completely, most likely due to a dox depot formation in vivo. The residual dox was sufficient to partly down-regulate a Tet-off controlled oncogene in a tumor transplantation experiment, resulting in reduced tumor growth. rtTA-CM2 mice may be a useful tool to analyze the function of genes in various organs but also reveal that down-regulation of gene expression is not complete.

D469del-COMP Retention in Chondrocytes Stimulates Caspase-Independent Necroptosis

Mutations in the cartilage oligomeric matrix protein gene (COMP) cause pseudoachondroplasia (PSACH). This dysplasia results from the intracellular retention of mutant COMP protein and premature death of growth-plate chondrocytes. Toward better understanding of these underlying mechanisms, we examined D469del-COMP activation of the unfolded protein response and cell death pathways in rat chondrosarcoma cells. Using an inducible expression system, we examined the effects of D469del-COMP retention after 4 days of mRNA expression and then 5 days without inducing agent. Retention of D469del-COMP stimulated Chop (Ddit3) and Gadd34 (Ppp1r15a) and triggered reactivation of protein translation that exacerbated intracellular retention. High levels of Nox4 and endoplasmic reticulum receptor stress-inducible Ero1β generated reactive oxygen species, causing oxidative stress. Increased expression of Gadd genes and presence of γH2AX indicated that DNA damage was occurring. The presence of cleaved apoptosis inducing factor (tAIF) and the absence of activated caspases indicated that retention of D469del-COMP triggers cell death in chondrocytes by necroptosis, a caspase-independent programmed necrosis. Loss of growth-plate chondrocytes by necroptosis was also found in our pseudoachondroplasia mouse model. These results suggest a model in which D469del-COMP expression induces persistent endoplasmic reticulum stress, oxidative stress, and DNA damage, thus priming chondrocytes for necroptosis. We define for the first time the precise mechanisms underlying D469del-COMP pathology in pseudoachondroplasia and suggest that oxidative stress and AIF may be promising therapeutic targets.

Gene Transfer Using HACs: A Key Step Closer to Ex Vivo Gene Therapy Using Autologous Gene–Corrected Cells to Treat Muscular Dystrophy

Gene Transfer Using HACs: A Key Step Closer to Ex Vivo Gene Therapy Using Autologous Gene–Corrected Cells to Treat Muscular Dystrophy

Doxycycline Regulatable Expression of Cytidine Deaminase Mediates Myeloprotection and Avoids Lymphotoxicity in a Murine Transplant Model

Abstract 2054Hematopoietic stem cells (HSCs) genetically modified to overexpress drug resistance genes have been advocated to overcome chemotherapy induced myelosuppression. In this context, we demonstrated that overexpression of hCDD from a constitutive spleen focus forming virus (SFFV)-derived promoter protects hematopoietic cells from Ara-C toxicity in vitro and in vivo. However, these studies also indicated substantial lymphotoxicity by high level constitutive CDD expression (Rattmann et al. Blood, 2006). To circumvent this problem, we now have established a Doxycycline (Dox)-inducible (TET-ON) CDD-expression system and have evaluated this system in murine in vitro and in vivo protection assays.In vitro CDD-mediated Ara-C resistance was evaluated in the hematopoietic cell line 32D as well as primary lineage negative (Lin-) clonogenic progenitor cells. In these studies cells were co-transduced with two lentiviral constructs expressing CDD (TET.CDD) or the reverse transactivator protein (rtTA3). In 32D cells, administration of 0.2 to 2.0 μg/ml Dox induced transgene expression to plateau levels within 24 hours. Rapid induction of the transgene also was observed at lower Dox concentrations, although exposure to 0.008 or 0.04 μg/ml led to notably reduced expression levels. Functionality of Dox-inducible hCDD expression was evaluated by exposing TET.CDD transgenic 32D cells to Ara-C. Transduced cells cultured in the presence of 2.0 μg/ml Dox proved completely resistant to Ara-C concentrations of up to 5000 nM, whereas eGFP- or not Dox treated control cells were susceptible to Ara-C exposure from 50 nM onwards. Again, hCDD-mediated drug resistance was dependent on the Dox dosage. When TET.CDD transduced 32D cells were exposed to Dox for 72h, marked protection from Ara-C was shown for Dox concentration of >0.2 μg/ml (LD50 > 2.000 nM Ara-C). After Dox withdrawal transgene expression remained detectable for at least three days. Similar protection was observed in primary hematopoietic cells and progenitor cell derived colonies were protected from Ara-C doses of 300 to 600 nM while untransduced control cells did not yield colony growth at doses of 50 nM Ara-C or higher.In vivo studies were performed by transplanting C57Bl/6 mice with Lin- cells from Rosa26-M2rtTA mice previously transduced with the TET.CDD or a control SIN lentiviral vector. Transgene expression was induced by Dox administration starting four weeks post transplantation. In this model Dox administration induced stable transgene expression in peripheral blood B, T, and myeloid cells peaking 15 days after start of administration and remaining detectable for 21 days after Dox withdrawal. No alterations in peripheral blood cell counts including the lymphocyte count were observed for up to eight weeks of Dox administration and also the relative contribution of gene modified cells to peripheral blood B, CD4+ or CD8+ T, and myeloid cells remained fairly constant during this time period. Even more important, a similar contribution of transduced cells was observed for the myeloid and the lymphoid cell compartment strongly arguing against a major lymphotoxicity. Also, no toxic effects of Dox-regulated hCDD expression was observed in other hematopoietic cell compartments including stem- or progenitor cells in various splenic, thymic and BM-derived hematopoietic cell compartments. Moreover, the TET.CDD vector conveyed significant protection against Ara-C (500 mg/kg, d1-4, i.v.) to the hematopoietic system as measured by granulocyte (0.26 +/−0.25 versus 0.8 +/−0, p=0.02) and platelet counts (584 +/−159 versus 883 +/−194, p=0.02) seven days after treatment. Furthermore, when the long term reconstitution potential of TET.CDD transduced Lin- cells was evaluated by secondary transplantation, robust, Dox-dependent transgenic eGFP expression was observed in peripheral blood B, CD4+ and CD8+ T, as well as myeloid cells of secondary recipients.Taken together, our data demonstrate efficient Dox-inducible hCDD expression in 32D and primary murine bone marrow cells in vitro as well as in a murine in vivo bone marrow transplant gene transfer model. Most importantly, in the latter model Dox-inducible CDD expression not only allowed for significant protection from Ara-C induced myelotoxicity but also abrogated the lymphotoxicity observed previously with high and constitutive hCDD expression. Disclosures:No relevant conflicts of interest to declare.

Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines

Lentiviral vectors (LVs) are considered one of the most promising vehicles to efficiently deliver genetic information for basic research and gene therapy approaches. Combining LVs with drug-inducible expression systems should allow tight control of transgene expression with minimal side effect on relevant target cells. A new doxycycline-regulated system based on the original TetR repressor was developed in 1998 as an alternative to the TetR-VP16 chimeras (tTA and rtTA) to avoid secondary effects due to the expression of transactivator domains. However, previously described TetR-based systems required cell cloning and/or antibiotic selection of tetracycline-responsive cells in order to achieve good regulation. In the present manuscript we have constructed a dual Tet-ON system based on two lentiviral vectors, one expressing the TetR through the spleen focus forming virus (SFFV) promoter (STetR) and a second expressing eGFP through the regulatable CMV-TetO promoter (CTetOE). Using these vectors we have demonstrated that the TetR repressor, contrary to the reverse transactivator (rtTA), can be expressed in excess to bind and modulate a high number of TetO operons. We have also showed that this dual vector system can generate regulatable bulk cell lines (expressing high levels of TetR) that are able to modulate transgene expression either by varying doxycycline concentration and/or by varying the amount of CTetOE vector genomes per cell. Based on these results we have developed a new all-in-one lentiviral vector (CEST) driving the expression of TetR through the SFFV promoter and the expression of eGFP through the doxycycline-responsive CMV-TetO operon. This vector efficiently produced Tet-ON regulatable immortalized (293T) and primary (human mesenchymal stem cells and human primary fibroblasts) cells. Bulk doxycycline-responsive cell lines express high levels of the transgene with low amount of doxycycline and are phenotypically indistinct from its parental cells.

A previously functional tetracycline-regulated transactivator fails to target gene expression to the bone

BackgroundThe tetracycline-controlled transactivator system is a powerful tool to control gene expression in vitro and to generate consistent and conditional transgenic in vivo model organisms. It has been widely used to study gene function and to explore pathological mechanisms involved in human diseases. The system permits the regulation of the expression of a target gene, both temporally and quantitatively, by the application of tetracycline or its derivative, doxycycline. In addition, it offers the possibility to restrict gene expression in a spatial fashion by utilizing tissue-specific promoters to drive the transactivator.FindingsIn this study, we report our problems using a reverse tetracycline-regulated transactivator (rtTA) in a transgenic mouse model system for the bone-specific expression of the Hutchinson-Gilford progeria syndrome mutation. Even though prior studies have been successful utilizing the same rtTA, expression analysis of the transactivator revealed insufficient activity for regulating the transgene expression in our system. The absence of transactivator could not be ascribed to differences in genetic background because mice in a mixed genetic background and in congenic mouse lines showed similar results.ConclusionsThe purpose of this study is to report our negative experience with previously functional transactivator mice, to raise caution in the use of tet-based transgenic mouse lines and to reinforce the need for controls to ensure the stable functionality of generated tetracycline-controlled transactivators over time.

Tetracycline‐regulated bone morphogenetic protein 2 gene expression in lentivirally transduced primary rabbit chondrocytes for treatment of cartilage defects

Treatment of cartilage defects is still challenging, primarily because of the poor self-healing capacity of articular cartilage. Gene therapy approaches have gained considerable attention, but, depending on the vector system used, they can lead to either limited or unrestrained gene expression, and therefore regulation of gene expression is necessary. This study was undertaken to construct an efficient tetracycline (Tet)-regulated, lentivirally mediated system for the expression of growth factor bone morphogenetic protein 2 (BMP-2) in primary rabbit chondrocytes that will allow for the induction and termination of growth factor gene expression once cartilage regeneration is complete. Chondrogenic ATDC5 cells and primary rabbit chondrocytes were lentivirally transduced with different tetracycline-on (Tet-On)-regulated, self-inactivating vectors for the induction of expression of enhanced green fluorescent protein (eGFP) or BMP-2, using either a 1-vector system or a 2-vector system. Expression of eGFP was induced on ATDC5 cells and chondrocytes. The highest induction rate and highest level of gene expression were reached when the spleen focus-forming virus long terminal repeat promoter was used to drive the reverse transactivator expression, after the addition of doxycycline, in chondrocytes. An up to 20-fold induction of Tet-mediated BMP-2 expression was observed on ATDC5 cells. The extent of induction and expression level of BMP-2 in chondrocytes were similar between the 1-vector system- and 2-vector system-infected cells (mean +/- SD 15.5 +/- 1.1 ng/ml and 14.6 +/- 0.4 ng/ml, respectively). In addition, prolonged induction and switching-off of BMP-2 expression, as well as repeated induction, were demonstrated. Production of proteoglycans, as shown by Alcian blue staining, demonstrated the functionality of the lentivirally expressed BMP-2 under induced conditions. The lentivirally mediated Tet-On system is an effective strategy for efficient, repeatedly inducible expression of BMP-2 in primary rabbit chondrocytes. Therefore, use of this system in in vivo experiments may be a promising approach as a treatment strategy for cartilage defects.

Anhydrotetracycline–peptide conjugates as representatives for ligand-based transactivating systems

Bioconjugates of anhydrotetracycline and minimal activation sequences (VP1, VP2) derived from the Herpes simplex virus protein VP16 were synthesized. Different ligation strategies were applied and the resulting molecules tested in HeLa cells expressing the reverse transactivator rtTA-S3 for activity. The data clearly demonstrate that the atc-peptide conjugates are able to penetrate the cell membrane. Furthermore, binding to and induction of rtTA-S3 were detected. Structure-activity relationships indicated that the biological activity of the atc-peptide strongly depends on the specific linker used. The N-terminally linked oxime derivative 10 proved excellent activity when the increase of luciferace activity indicated a transcriptional activation substantially exceeding the inducing properties of anhydrotetracycline.

Adjusting transgene expression levels in lymphocytes with a set of inducible promoters

Inducible gene expression systems are powerful research tools and could be of clinical value in the future, with lymphocytes being likely prime application targets. However, currently available regulatable promoters exhibit variation in their efficiency in a cell line-dependent-manner and are notorious for basal leakiness or poor inducibility. Data concerning the regulatory properties of different inducible promoters are scarce for lymphocytes. In the present study, we report a comprehensive analysis of how various inducible promoters perform and how their combination with a transsilencer and a reverse transactivator can result in optimally controlled gene expression in T-cells. The performance of the tetracycline-regulated (Tet)-inducible promoters Tet-responsive element (TRE), mouse mammary tumor virus (MMTV)/TRE, TREtight and second generation TRE (SG/TRE) was compared in several B-cell lines and in Jurkat T-cells using transient transfections in combination with Tet-On. To monitor transgene expression in a Jurkat cell line containing a transsilencer and a reverse transactivator, expression cassettes encoding enhanced green fluorescent protein, CD123 or a constitutively active, cytotoxic caspase-3 were flanked with insulators and stably integrated. The performance of TREtight and SG/TRE was furthermore analysed in transiently transfected primary CD4(+) human T-cells. The promoters exhibit greatly diverging characteristics. MMTV/TRE permits moderate, TRE and TREtight permits intermediate and SG/TRE permits very high expression levels. TRE and SG/TRE are leaky, whereas MMTV/TRE and TREtight provide stringent expression control. Tetracycline derivatives add flexibility to transgene expression by introducing intermediate expression levels. The different expression profiles of the promoters increase the flexibility to adjust transgene expression levels. The promoters provide an additional option to optimize system performance for many applications.