Tetracycline-regulated System Research Articles

Using recombinase-mediated cassette exchange to engineer MIN6 insulin-secreting cells based on a newly identified safe harbor locus.

Aims/IntroductionRecent studies have identified genomic and transcript level changes along with alterations in insulin secretion in patients with diabetes and in rodent models of diabetes. It is important to establish an efficient system for testing functional consequences of these changes. We aimed to generate such a system using insulin‐secreting MIN6 cells.Materials and MethodsMIN6 cells were first engineered to have a tetracycline‐regulated expression system. Then, we used the recombination‐mediated cassette exchange strategy to explore the silencing‐resistant site in the genome and generated a master cell line based on this site.ResultsWe identified a site 10.5 kbps upstream from the Zxdb gene as a locus that allows homogenous transgene expression from a tetracycline responsible promoter. Placing the Flip/Frt‐based platform on this locus using CRISPR/Cas9 technology generated modified MIN6 cells applicable to achieving cassette exchange on the genome. Using this cell line, we generated MIN6 subclones with over‐ or underexpression of glucokinase. By analyzing a mixed population of these cells, we obtained an initial estimate of effects on insulin secretion within 6 weeks. Furthermore, we generated six MIN6 cell sublines simultaneously harboring genes of inducible overexpression with unknown functions in insulin secretion, and found that Cited4 and Arhgef3 overexpressions increased and decreased insulin secretion, respectively.ConclusionsWe engineered MIN6 cells, which can serve as a powerful tool for testing genetic alterations associated with diabetes, and studied the molecular mechanisms of insulin secretion.

Analysis of the Contribution of NF-κB in the Regulation of Chemotherapy-Induced Cell Senescence by Establishing a Tetracycline-Regulated Cell System.

Therapy-induced senescence (TIS or therapy-induced premature senescence) is a key cellular program triggered in the course of cancer radiotherapy and chemotherapy with genotoxic drugs, both in cancer cells and in normal cells, whose activation critically affects the outcome of cancer therapy. Drug-induced senescent cells undergo a permanent cell cycle arrest, acquire distinctive morphological and biochemical alterations, and an enhanced secretory ability, referred to as senescence-associated secretory phenotype (SASP). The transcription factor NF-κB acts as a master regulator of the SASP, driving the expression of senescence-associated secretome components.Here we describe protocols for the establishment of a tetracycline-regulated cell system for the investigation of the role of NF-κB in TIS. We also describe protocols routinely used in our laboratory, to investigate TIS in this Tet-On inducible expression system. Finally, we describe techniques for the validation of TIS induction.

Candida albicans Aro1 affects cell wall integrity, biofilm formation and virulence.

Candida albicans is an opportunistic pathogen capable of causing life-threatening systemic infections. The C. albicans ARO1 gene encodes an arom multifunctional enzyme, which can possibly catalyze reactions of the shikimate pathway to synthesize aromatic amino acids. However, the functions of C. albicans Aro1 have not been extensively characterized. ARO1 knockdown mutant strain was constructed, using a tetracycline-regulated (TR) expression system. Cell growth of the mutant strain was compared with wild type. Effects of the ARO1 gene knockdown on cell wall properties, adhesion to polystyrene and biofilm formation were further investigated. Finally, Galleria mellonella was used as a model host to study the role of ARO1 in virulence of C. albicans. We showed that defective growth in the ARO1 knockdown strain was rescued by supplemental aromatic amino acids. In addition, the ARO1 knockdown strain was easily aggregated and precipitated. The knockdown of ARO1 also caused changes in cell wall properties and compositions and promoted C. albicans cell adhesion to polystyrene and biofilm formation. Finally, the ARO1 knockdown strain showed attenuation of C. albicans virulence. This work provides new insights into C. albicans metabolism, cell wall and virulence.

Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors.

PurposeThe cyclin-dependent kinase 1 (Cdk1) and cyclin B complex performs important roles in the transition from the G2 to M phase in the cell cycle through removal of inhibitory phosphates on Cdk1, and Cdc25B, which is a dual-specific phosphatase, mediates these dephosphorylation events. However, measuring Cdc25B activity by existing methods is hampered by inadequate nonspecific substrates and the need to use a radiolabeled isotope. The present study aimed to develop an improved method with which to properly measure Cdc25B activity using a novel nonradioisotopic assay and Cdc25B overexpression cell lines.Materials and MethodsA nonradioisotopic Cdk1 kinase assay, based on Western blotting for retinoblastoma protein and histone H1, was used to analyze Cdc25B activity. Also, stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines were constructed using the tetracycline-regulated expression system and were applied as a tool for screening for inhibitors of Cdc25B.ResultsThe present study developed and optimized a nonradioisotopic assay method to properly measure Cdc25B activity. Furthermore, we constructed stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines for the establishment of a strong assay system with which to evaluate the specificity of Cdc25B inhibitors under conditions similar to the intracellular environment. These methods were confirmed as useful tools for measuring Cdc25B activity.ConclusionThe nonradioisotopic Cdk1 kinase assay and Cdc25B overexpression cell lines developed in this study can be conveniently used as tools for screening inhibitors of Cdc25B phosphatase as anticancer drugs.

ApoL1 Overexpression Drives Variant-Independent Cytotoxicity.

Coding variants in the APOL1 gene are associated with kidney diseases in African ancestral populations; yet, the underlying biologic mechanisms remain uncertain. Variant-dependent autophagic and cytotoxic cell death have been proposed as pathogenic pathways mediating kidney injury. To examine this possibility, we conditionally expressed APOL1-G0 (reference), -G1, and -G2 (variants) using a tetracycline-regulated system in HEK293 cells. Autophagy was monitored biochemically and cell death was measured using multiple assays. We measured intracellular Na+ and K+ content with atomic absorption spectroscopy and APOL1-dependent currents with whole-cell patch clamping. Neither reference nor variant APOL1s induced autophagy. At high expression levels, APOL1-G0, -G1, and -G2 inserted into the plasma membrane and formed pH-sensitive cation channels, causing collapse of cellular Na+ and K+ gradients, phosphorylation of p38 mitogen-activated protein kinase, and cell death, without variant-dependent differences. APOL1-G0 and -G2 exhibited similar channel properties in whole-cell patch clamp experiments. At low expression levels, neither reference nor variant APOL1s localized on the plasma membrane, Na+ and K+ gradients were maintained, and cells remained viable. Our results indicate that APOL1-mediated pore formation is critical for the trypanolytic activity of APOL1 and drives APOL1-mediated cytotoxicity in overexpression systems. The absence of cytotoxicity at physiologic expression levels suggests variant-dependent intracellular K+ loss and cytotoxicity does not drive kidney disease progression.

Fusion of Human Fetal Mesenchymal Stem Cells with "Degenerating" Cerebellar Neurons in Spinocerebellar Ataxia Type 1 Model Mice.

Mesenchymal stem cells (MSCs) migrate to damaged tissues, where they participate in tissue repair. Human fetal MSCs (hfMSCs), compared with adult MSCs, have higher proliferation rates, a greater differentiation capacity and longer telomeres with reduced senescence. Therefore, transplantation of quality controlled hfMSCs is a promising therapeutic intervention. Previous studies have shown that intravenous or intracortical injections of MSCs result in the emergence of binucleated cerebellar Purkinje cells (PCs) containing an MSC-derived marker protein in mice, thus suggesting a fusion event. However, transdifferentiation of MSCs into PCs or transfer of a marker protein from an MSC to a PC cannot be ruled out. In this study, we unequivocally demonstrated the fusion of hfMSCs with murine PCs through a tetracycline-regulated (Tet-off) system with or without a Cre-dependent genetic inversion switch (flip-excision; FLEx). In the FLEx-Tet system, we performed intra-cerebellar injection of viral vectors expressing tetracycline transactivator (tTA) and Cre recombinase into either non-symptomatic (4-week-old) or clearly symptomatic (6–8-month-old) spinocerebellar ataxia type 1 (SCA1) mice. Then, the mice received an injection of 50,000 genetically engineered hfMSCs that expressed GFP only in the presence of Cre recombinase and tTA. We observed a significant emergence of GFP-expressing PCs and interneurons in symptomatic, but not non-symptomatic, SCA1 mice 2 weeks after the MSC injection. These results, together with the results obtained using age-matched wild-type mice, led us to conclude that hfMSCs have the potential to preferentially fuse with degenerating PCs and interneurons but not with healthy neurons.

Terminal differentiation induction as DNA damage response in hematopoietic stem cells by GADD45A.

Hematopoietic stem cells (HSCs) sustain lifelong blood cell regeneration by balancing their ability for self-renewal with their ability to differentiate into all blood cell types. To prevent organ exhaustion and malignant transformation, long-lived HSCs, in particular, must be protected from exogenous and endogenous stress, which cause severe DNA damage. When DNA is damaged, distinct DNA repair mechanisms and cell fate controls occur in adult HSCs compared with committed cells. Growth arrest and DNA damage-inducible 45 alpha (GADD45A) is known to coordinate a variety of cellular stress responses, indicating the molecule is an important stress mediator. So far, the function of GADD45A in hematopoietic stem and progenitor cells is controversial and appears highly dependent on the cell type and stress stimulus. Recent studies have analyzed its role in cell fate decision control of prospectively isolated HSCs and have revealed unexpected functions of GADD45A, as discussed here. The upregulation of GADD45A by DNA damage-causing conditions results in enhanced HSC differentiation, probably to efficiently eliminate aberrant HSCs from the system. These findings, in concert with a few studies on other stem cell systems, have led us to propose DNA damage-induced differentiation as a novel DNA damage response mechanism in stem cells that circumvents the fatal consequences of cumulative DNA damage in the stem cell compartment.

Cellular Dynamics of Memory B Cell Populations: IgM+ and IgG+ Memory B Cells Persist Indefinitely as Quiescent Cells.

Despite their critical role in long-term immunity, the life span of individual memory B cells remains poorly defined. Using a tetracycline-regulated pulse-chase system, we measured population turnover rates and individual t1/2 of pre-established Ag-induced Ig class-switched and IgM-positive memory B cells over 402 d. Our results indicate that, once established, both IgG-positive and less frequent IgM-positive memory populations are exceptionally stable, with little evidence of attrition or cellular turnover. Indeed, the vast majority of cells in both pools exhibited t1/2 that appear to exceed the life span of the mouse, contrasting dramatically with mature naive B cells. These results indicate that recall Ab responses are mediated by stable pools of extremely long-lived cells, and suggest that Ag-experienced B cells employ remarkably efficient survival mechanisms.

The generation of a ZBTB16-inducible expression system in the ACHN adenocarcinoma cell line

Abstract Objectives To establish an inducible system for controlled ZBTB16 protein expression in a renal adenocarcinoma cell line. Methods The Tet-On ® 3G Inducible Expression System from Clontech was used to generate a tetracycline-regulated ZBTB16 expression system in the ACHN renal adenocarcinoma cell line. Results The Tet-On ® 3G system successfully controlled the basal level of ZBTB16 with no detectable protein expression when doxycycline was absent from the culture medium. The addition of doxycycline induced ZBTB16 protein expression with as low as 20 ng/ml of doxycycline. Conclusion The firm regulation of transcription factor expression in cancer cell models is very important in investigating their biological roles. Many transcription factors, such as ZBTB16, will carry on their biological activities even with minimal expression levels. The Tet-On ® 3G system was essential in controlling the basal expression to prevent transcription leakage and providing a tightly regulated method for expressing ZBTB16 in a manner resembling physiological conditions.

Gene transfer engineering for astrocyte-specific silencing in the CNS.

Cell-type-specific gene silencing is critical to understand cell functions in normal and pathological conditions, in particular in the brain where strong cellular heterogeneity exists. Molecular engineering of lentiviral vectors has been widely used to express genes of interest specifically in neurons or astrocytes. However, we show that these strategies are not suitable for astrocyte-specific gene silencing due to the processing of small hairpin RNA (shRNA) in a cell. Here we develop an indirect method based on a tetracycline-regulated system to fully restrict shRNA expression to astrocytes. The combination of Mokola-G envelope pseudotyping, glutamine synthetase promoter and two distinct microRNA target sequences provides a powerful tool for efficient and cell-type-specific gene silencing in the central nervous system. We anticipate our vector will be a potent and versatile system to improve the targeting of cell populations for fundamental as well as therapeutic applications.

598. Fusion of Mesenchymal Stem Cells Exclusively with “Degenerating” Cerebellar Neurons in Spinocerebellar Ataxia Type 1 Model Mice

Mesenchymal stem cells (MSCs) have the potential to migrate to damaged tissues where they participate in tissue repair. Previous studies suggest that intravenous or intracortical injections of MSCs results in the emergence of binucleated cerebellar Purkinje cells (PCs) containing a MSC-derived marker protein in mice suggestive of a fusion event. However, transdifferentiation of MSCs into PCs or transfer of a marker protein from a MSC to a PC cannot be completely ruled-out. Here we unequivocally showed a fusion of human fetal MSCs (hMSCs) with mouse PCs by combination of tetracycline-regulated system with Cre-dependent genetic inversion switch (flip-excision; FLEx). We performed intra-cerebellar injection of adeno-associated viral vectors expressing tetracycline transactivator (tTA) and Cre recombinase into either non-symptomatic (4 week-old) or clearly symptomatic (6 – 8 month-old) spinocerebellar ataxia type 1 (SCA1) mice. Two weeks after the treatment, the mice received injection of 50,000 geneticially-engineered hMSCs that expressed GFP only in the presence of Cre recombinase and tTA, which restricted GFP expression exclusively to cerebellar cells fused with hMSCs. We observed GFP-expressing PCs and interneurons in symptomatic, but not non-symptomatic, SCA1 mice as early as 2 weeks after the MSC injection. The frequency of GFP-expressing PCs did not increase when analysed at longer time points (≈23 weeks post hMSC grafting), suggesting that most hMSC fusion events occurred within 2 weeks of delivery. Using this model, we show conclusively that hMSC display a tendency to fuse with degenerating PC and interneurons, and not with normal neurons.

Id3 induces an Elk-1-caspase-8-dependent apoptotic pathway in squamous carcinoma cells.

Inhibitor of differentiation/DNA-binding (Id) proteins are helix–loop–helix (HLH) transcription factors. The Id protein family (Id1–Id4) mediates tissue homeostasis by regulating cellular processes including differentiation, proliferation, and apoptosis. Ids typically function as dominant negative HLH proteins, which bind other HLH proteins and sequester them away from DNA promoter regions. Previously, we have found that Id3 induced apoptosis in immortalized human keratinocytes upon UVB exposure, consistent with its role as a tumor suppressor. To investigate the role of Id3 in malignant squamous cell carcinoma (SCC) cells (A431), a tetracycline-regulated inducible system was used to induce Id3 in cell culture and mouse xenograft models. We found that upon Id3 induction, there was a decrease in cell number under low serum conditions, as well as in soft agar. Microarray, RT-PCR, immunoblot, siRNA, and inhibitor studies revealed that Id3 induced expression of Elk-1, an E-twenty-six (ETS)-domain transcription factor, inducing procaspase-8 expression and activation. Id3 deletion mutants revealed that 80 C-terminal amino acids, including the HLH, are important for Id3-induced apoptosis. In a mouse xenograft model, Id3 induction decreased tumor size by 30%. Using immunofluorescent analysis, we determined that the tumor size decrease was also mediated through apoptosis. Furthermore, we show that Id3 synergizes with 5-FU and cisplatin therapies for nonmelanoma skin cancer cells. Our studies have shown a molecular mechanism by which Id3 induces apoptosis in SCC, and this information can potentially be used to develop new treatments for SCC patients.

A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expression

BackgroundOne of the most commonly used vectors for gene therapy is the adenoviral vector; its ability to tightly regulate transgene expression is critical for optimizing therapeutic outcomes. The tetracycline-regulated system (especially the Tet-On system) for gene expression is one of the most valuable tools for controlling gene expression. The major problem of an adenoviral vector carrying a Tet-On system is suboptimal regulation of transgene expression.ResultsWe constructed a single adenoviral vector carrying in its E1 region a novel “all-in-one” Tet-On system with an autoregulatory loop. This system had improved Dox-inducible gene expression in terms of low basal expression, high induced expression and high responsiveness to Dox. To our knowledge, this is the first reported adenovirus-based, all-in-one Tet-On system with an autoregulatory loop inserted into a single region of adenoviral genome. This system was further tested by inducible expression of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). The adenovirus that expressed soluble TRAIL under the control of this novel Tet-On system showed tumor-derived cells inhibitory activity in SW480 cells only under induced conditions.ConclusionsOur novel, single adenoviral vector carrying in its E1 region an all-in-one Tet-On system with an autoregulatory loop displayed tight regulation of transgene expression in vitro. This system has great potential for a variety of applications, including gene therapy and the study of gene function.

Calreticulin Regulates VEGF-A in Neuroblastoma Cells.

Calreticulin (CRT) has been previously correlated with the differentiation of neuroblastoma (NB), implying a favorable prognostic factor. Vascular endothelial growth factor (VEGF) has been reported to participate in the behavior of NB. This study investigated the association of CRT and VEGF-A in NB cells. The expressions of VEGF-A and HIF-1α, with overexpression or knockdown of CRT, were measured in three NB cells (SH-SY5Y, SK-N-DZ, and stNB-V1). An inducible CRT NB cell line and knockdown CRT stable cell lines were also established. The impacts of CRT overexpression on NB cell apoptosis, proliferation, and differentiation were also evaluated. We further examined the role of VEGF-A in the NB cell differentiation via VEGF receptor blockade. Constitutive overexpression of CRT led to NB cell differentiation without proliferation. Thus, an inducible CRT stNB-V1 cell line was generated by a tetracycline-regulated gene system. CRT overexpression increased VEGF-A and HIF-1α messenger RNA (mRNA) expressions in SH-SY5Y, SK-N-DZ, and stNB-V1 cells. CRT overexpression also enhanced VEGF-A protein expression and secretion level in conditioned media in different NB cell lines. Knockdown of CRT decreased VEGF-A and HIF-1α mRNA expressions and lowered VEGF-A protein expression and secretion level in conditioned media in different NB cell lines. We further demonstrated that NB cell apoptosis was not affected by CRT overexpression in stNB-V1 cells. Nevertheless, overexpression of CRT suppressed cell proliferation and enhanced cell differentiation in stNB-V1 cells, whereas blockage of VEGFR-1 markedly suppressed the expression of neuron-specific markers including GAP43, NSE2, and NFH, as well as TrkA, a molecular marker indicative of NB cell differentiation. Our findings suggest that VEGF-A is involved in CRT-related neuronal differentiation in NB. Our work may provide important information for developing a new therapeutic strategy to improve the outcome of NB patients.

Conditional expression of the type 2 angiotensin II receptor in mesenchymal stem cells inhibits neointimal formation after arterial injury.

Percutaneous coronary interventions (PCIs) are an effective treatment for obstructive coronary artery diseases. However, the procedure's success is limited by remodeling and formation of neointima. In the present study, we engineered rat mesenchymal stem cells (MSCs) to express type 2 angiotensin II receptor (AT2R) using a tetracycline-regulated system that can strictly regulate AT2R expression. We tested the ability of the modified MSCs to reduce neointima formation following arterial injury. We subjected rats to balloon injury, and reverse transcriptase polymerase chain reaction (RT-PCR) indicated no significant AT2R expression in normal rat arteries. Low expression of AT2R was observed at 28 days after balloon-induced injury. Interestingly, MSCs alone were unable to reduce neointimal hyperplasia after balloon-induced injury; after transplantation of modified MSCs, doxycycline treatment significantly upregulated neointimal AT2R expression and inhibited osteopontin mRNA expression, as well as neointimal formation. Taken together, these results suggest that transplantation of MSCs conditionally expressing AT2R could effectively suppress neointimal hyperplasia following balloon-induced injury. Therefore, MSCs with a doxycycline-controlled gene induction system may be useful for the management of arterial injury after PCI.

Development of a multipurpose GATEWAY-based lentiviral tetracycline-regulated conditional RNAi system (GLTR).

RNA interference (RNAi) has become an essential technology for functional gene analysis. Its success, however, depends on the effective expression of RNAi-inducing small double-stranded interfering RNA molecules (siRNAs) in target cells. In many cell types, RNAi can be achieved by transfection of chemically synthesised siRNAs, which results in transient knockdown of protein expression. Expression of double-stranded short hairpin RNA (shRNA) provides another means to induce RNAi in cells that are hard to transfect. To facilitate the generation of stable, conditional RNAi cell lines, we have developed novel one- and two-component vector GATEWAY-compatible lentiviral tetracycline-regulated RNAi (GLTR) systems. The combination of a modified RNA-polymerase-III-dependent H1 RNA promoter (designated ‘THT’) for conditional shRNA expression with different lentiviral delivery vectors allows (1) the use of fluorescent proteins for colour-coded combinatorial RNAi or for monitoring RNAi induction (pGLTR-FP), (2) selection of transduced cells (pGLTR-S), and (3) the generation of conditional cell lines using a one vector system (pGLTR-X). All three systems were found to be suitable for the analysis of essential genes, such as CDC27, a component of the mitotic ubiquitin ligase APC/C, in cell lines and primary human cells.

Spatiotemporal control of porcine p65RHD expression by advanced Tet-On system in PIEC cells helps regulate NFкB activity

NFкB transcription activation leads to malfunction of endothelial cells, which is the main reason for pig xenograft rejection. Overexpression of a dominant negative mutant of porcine NFκB p65 (pp65RHD) could inhibit NFкB activation in endothelial cells. This study presents an advanced tetracycline-regulated system for pp65RHD spatiotemporal expression in porcine iliac endothelial cell line. In this system, an endothelial specific promoter ICAM-2 is used to improve pTet-On and internal ribosome entry site as well as enhanced green fluorescent protein (EGFP) elements are used to facilitate the result observation in pTRE-Tight. Through transfection and drug selection, we obtained 7 single cell clones containing the advanced Tet-On system, in which pp65RHD expression is under tight regulated by doxycycline and can be visualized easily through EGFP. The distribution of induced pp65RHD was verified by immunocytochemical assays test. Then, NFкB activity was tested. Luciferase reporter assays showed that NFкB activity in two clones was influenced by the Dox-induced pp65RHD expression, but other clones weren't influenced. Therefore, we picked up 2 cell clones from the uninfluenced clones for further investigation by immunocytochemical assays and RT-PCR detection. The final results supported the overexpression of pp65RHD in one clone could successfully inhibit NFкB activity. The success of pp65RHD spatiotemporal expression system is helpful to regulate NFкB activity and conquer cell-mediated immunity and could be used for preparation of transgenic pig, contributing to xenotransplantation.

Functional coupling of chloride–proton exchanger ClC-5 to gastric H+,K+-ATPase

SummaryIt has been reported that chloride–proton exchanger ClC-5 and vacuolar-type H+-ATPase are essential for endosomal acidification in the renal proximal cells. Here, we found that ClC-5 is expressed in the gastric parietal cells which secrete actively hydrochloric acid at the luminal region of the gland, and that it is partially localized in the intracellular tubulovesicles in which gastric H+,K+-ATPase is abundantly expressed. ClC-5 was co-immunoprecipitated with H+,K+-ATPase in the lysate of tubulovesicles. The ATP-dependent uptake of 36Cl− into the vesicles was abolished by 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH28080), an inhibitor of H+,K+-ATPase, suggesting functional expression of ClC-5. In the tetracycline-regulated expression system of ClC-5 in the HEK293 cells stably expressing gastric H+,K+-ATPase, ClC-5 was co-immunoprecipitated with H+,K+-ATPase, but not with endogenous Na+,K+-ATPase. The SCH28080-sensitive 36Cl− transporting activity was observed in the ClC-5-expressing cells, but not in the ClC-5-non-expressing cells. The mutant (E211A-ClC-5), which has no H+ transport activity, did not show the SCH28080-sensitive 36Cl− transport. On the other hand, both ClC-5 and its mutant (E211A) significantly increased the activity of H+,K+-ATPase. Our results suggest that ClC-5 and H+,K+-ATPase are functionally associated and that they may contribute to gastric acid secretion.

Construction of a Single Lentiviral Vector Containing Tetracycline-Inducible Alb-uPA for Transduction of uPA Expression in Murine Hepatocytes

The SCID-beige/Alb-uPA mouse model is currently the best small animal model available for viral hepatitis infection studies [1]. But the construction procedure is often costly and time-consuming due to logistic and technical difficulties. Thus, the widespread application of these chimeric mice has been hampered [2]. In order to optimize the procedure, we constructed a single lentiviral vector containing modified tetracycline-regulated system to control Alb-uPA gene expression in the cultured hepatocytes. The modified albumin promoter controlled by tetracycline (Tet)-dependent transactivator rtTA2S-M2 was integrated into a lentiviral vector. The full-length uPA cDNA was inserted into another lentiviral vector containing PTight, a modified Tet-responsive promoter. Two vectors were then digested by specific enzymes and ligated by DNA ligase 4. The ligated DNA fragment was inserted into a modified pLKO.1 cloning vector and the final lentiviral vector was then successfully constructed. H2.35 cell, Lewis lung carcinoma, primary kidney, primary hepatic interstitial and CT26 cells were infected with recombinant lentivirus at selected MOI. The expression of uPA induced by DOX was detectable only in the infected H2.35 cells, which was confirmed by real-time PCR and Western blot analysis. Moreover, DOX induced uPA expression on the infected H2.35 cells in a dose-dependent manner. The constructed single lentiviral vector has many biological advantages, including that the interested gene expression under “Tet-on/off” system is controlled by DOX in a dose-depending fashion only in murine liver cells, which provides an advantage for simplifying generation of conditional transgenic animals.

Sox21 inhibits glioma progressionin vivoby forming complexes with Sox2 and stimulating aberrant differentiation

Sox2 is a transcription factor in neural stem cells and keeps the cells immature and proliferative. Sox2 is expressed in primary human glioma such as glioblastoma multiforme (GBM), primary glioma cells and glioma cell lines and is implicated in signaling pathways in glioma connected to malignancy. Sox21, the counteracting partner of Sox2, has the same expression pattern as Sox2 in glioma but in general induces opposite effects. In this study, Sox21 was overexpressed by using a tetracycline-regulated expression system (tet-on) in glioma cells. The glioma cells were injected subcutaneously into immunodeficient mice. The control tumors were highly proliferative, contained microvascular proliferation and large necrotic areas typical of human GBM. Induction of Sox21 in the tumor cells resulted in a significant smaller tumor size, and the effect correlated with the onset of treatment, where earlier treatment gave smaller tumors. Mice injected with glioma cells orthotopically into the brain survived significantly longer when Sox21 expression was induced. Tumors originating from glioma cells with an induced expression of Sox21 exhibited an increased formation of Sox2:Sox21 complexes and an upregulation of S100β, CNPase and Tuj1. Sox21 appears to decrease the stem-like cell properties of the tumor cells and initiate aberrant differentiation of glioma cells in vivo. Taken together our results indicate that Sox21 can function as a tumor suppressor during gliomagenesis mediated by a shift in the balance between Sox2 and Sox21. The wide distribution of Sox2 and Sox21 in GBM makes the Sox2/Sox21 axis a very interesting target for novel therapy of gliomas.