Alkaline Phosphatase Reporter Gene Research Articles

Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A1 receptor ligands

Background and purpose:The introduction of fluorescence-based techniques, and in particular the development of fluorescent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level. This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties of fluorescent agonists and antagonists.Experimental approach:Chinese hamster ovary cells stably expressing the human adenosine A1 receptor and a cyclic 3′,5′ adenosine monophosphate response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene, together with whole cell [3H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX) radioligand binding, were used to evaluate the pharmacological properties of a range of fluorescent ligands based on the antagonist xanthine amine congener (XAC) and the agonist 5′ (N-ethylcarboxamido) adenosine (NECA).Key results:Derivatives of NECA and XAC with different fluorophores, but equivalent linker length, showed significant differences in their binding properties to the adenosine A1 receptor. The BODIPY 630/650 derivatives had the highest affinity. Linker length also affected the pharmacological properties, depending on the fluorophore used. Particularly in fluorescent agonists, higher agonist potency could be achieved with large or small linkers for dansyl and BODIPY 630/650 derivatives, respectively.Conclusions and implications:The pharmacology of a fluorescent ligand was critically influenced by both the fluorophore and the associated linker. Furthermore, our data strongly suggest that the physicochemical properties of the fluorophore/linker pairing determine where in the environment of the target receptor the fluorophore is placed, and this, together with the environmental sensitivity of the resulting fluorescence, may finally decide its utility as a fluorescent probe.This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x

T.P.6.06 Real-time monitoring of cell transplantation in mouse dystrophic muscles by a secreted alkaline phosphatase reporter gene

T.P.6.06 Real-time monitoring of cell transplantation in mouse dystrophic muscles by a secreted alkaline phosphatase reporter gene

Real-time monitoring of cell transplantation in mouse dystrophic muscles by a secreted alkaline phosphatase reporter gene

Transplantation of muscle precursor cells (MPCs) is a promising approach for the treatment of muscular dystrophies. However, preclinical and clinical results have shown that the technology is not yet efficient enough for most therapeutic applications. Among the problems that remain unsolved are low cellular survival, poor proliferation and lack of migration of the transplanted cells. One major technical hurdle for the optimization of transplantation protocols is how to follow precisely the fate of the cells after transplantation. In this study, we examined the use of a secreted form of the mouse alkaline phosphatase (mSeAP) enzyme as the reporter system transduced into MPCs using a retroviral vector. We show that circulating mSeAP could be detected in the serum of the transplanted mice at different time points after MPC transplantation. We also found that the level of circulating mSeAP is highly correlated with the number of transplanted cells and that mSeAP is an excellent histological marker. Further, studying the levels of circulating mSeAP compared with the number of muscle fibers positive to mSeAP and to dystrophin, enabled detailed analyses of bottleneck steps for successful transplantation. Taken together, our results show that mSeAP is an excellent quantitative 'real-time' reporter gene for cell therapy preclinical studies.

A Single Intravenous Injection of Adeno-associated Virus Serotype-9 Leads to Whole Body Skeletal Muscle Transduction in Dogs

The success of many gene therapy applications hinges on efficient whole body transduction. In the case of muscular dystrophies, a therapeutic vector has to reach every muscle in the body. Recent studies suggest that vectors based on adeno-associated virus (AAV) are capable of body-wide transduction in rodents. However, translating this finding to large animals remains a challenge. Here we explored systemic gene delivery with AAV serotype-9 (AAV-9) in neonatal dogs. Previous attempts to directly deliver AAV to adult canine muscle have yielded minimal transduction due to a strong cellular immune response. However, in neonatal dogs we observed robust skeletal muscle transduction throughout the body after a single intravenous injection. Importantly, systemic transduction was achieved in the absence of pharmacological intervention or immune suppression and it lasted for at least 6 months (the duration of study). We also observed several unique features not predicted by murine studies. In particular, cardiac muscle was barely transduced in dogs. Many muscular dystrophy patients can be identified by neonatal screening. The technology described here may lead to an effective early intervention in these patients.

Amantadine as a regulator of internal ribosome entry site

Studies of eukaryotes have yielded 2 translation initiation mechanisms: a classical cap-dependent mechanism and a cap-independent mechanism proceeding through the internal ribosomal entry site (IRES). We hypothesized that it might be possible to identify compounds that may distinguish between cap-dependent translation and cap-independent IRES-mediated translation. To facilitate compound screening, we developed bicistronic reporter constructs containing a beta-galactosidase gene (beta-gal) and a secreted human placental alkaline phosphatase (SEAP) reporter gene. Following transcription, the beta-gal gene is translated by a cap-dependent mechanism, while SEAP expression is controlled by the IRES derived from either enterovirus 71 (EV-71) or encephalomyocarditis virus (EMCV). This assay could potentially identify compounds that inhibit SEAP expression (cap-independent) without affecting beta-gal activity (cap-dependent). Using a bicistronic plasmid-based transient transfection assay in the COS-1 cells, we identified amantadine, a compound that inhibited the IRES of EV71- and EMCV-mediated cap-independent translation but did not interfere with cap-dependent translation when the dose of amantadine was lower than 0.25 mg/mL. These results imply that amantadine may distinguish between cap-dependent translation and cap-independent IRES-mediated translation and can be used to regulate gene expression at a translational level.

Measurement of the Humoral Immune Response following an Incident Human Papillomavirus Type 16 or 18 Infection in Young Women by a Pseudovirion-Based Neutralizing Antibody Assay

We have evaluated a neutralizing antibody assay which uses human papillomavirus (HPV) type 16 (HPV-16) and HPV-18 pseudovirions carrying a secretory alkaline phosphatase reporter gene and which can potentially measure functionally relevant HPV type-specific neutralizing antibodies. The reproducibility of the assay was excellent; for HPV-16, the intra- and interassay kappa values were 0.95 and 0.90, respectively; and for HPV-18, the corresponding values were 0.90 and 0.90. This assay was used to describe the kinetics of the neutralizing antibody response in a cohort of 42 young women who were recruited soon after first intercourse and who first tested positive for HPV-16 DNA or HPV-18 DNA, or both, during follow-up. Most women seroconverted following the first detection of type-specific HPV DNA and remained seropositive until the end of follow-up. Our findings are broadly consistent with those of two other cohort studies which have measured the serological response following an incident infection by using the technically simpler virus-like-particle-based enzyme-linked immunosorbent assay.

Progesterone receptor antagonists with a 3-phenylquinazoline-2,4-dione/2-phenylisoquinoline-1,3-dione skeleton

Novel non-steroidal progesterone receptor antagonists with a 3-phenylquinazoline-2,4-dione/2-phenylisoquinoline-1,3-dione skeleton were developed and their structure–activity relationships were investigated. Among the prepared compounds, 4-(4,4-diethyl-3,4-dihydro-1,3-dioxoquinolin-2(1 H)-yl)benzonitrile (DEPIQ-4CN) showed the most potent activity, with IC 50 values of 74–78 nM in alkaline phosphatase activity and reporter gene assays.

Sustained liver‐specific transgene expression from the albumin promoter in mice following hydrodynamic plasmid DNA delivery

To properly study gene expression in vivo, often long-term expression is desired. Previous studies using plasmid DNA (pDNA) vectors have typically resulted in short-term expression. Here, we evaluated combinations of the albumin promoter with different enhancers and untranslated regions for liver-specific expression in mice. A series of pDNA secreted alkaline phosphatase (SEAP) reporter gene expression vectors was constructed using the albumin promoter and various other expression cassette elements. Each was evaluated for level and duration of SEAP expression in mice following hydrodynamic tail vein delivery. Sustained liver expression was obtained from vectors combining the albumin promoter with an albumin 3' untranslated region (3'UTR). The level of expression was increased by inclusion of enhancers and a 5' intron. The optimal expression vector consisted of the albumin promoter combined with an alpha-fetoprotein MERII enhancer, 5' intron from the factor IX gene, and the 3'UTR from the albumin gene including intron 14. With this vector, SEAP reporter gene expression levels remained high for 1 year, at levels comparable to those obtained from the cytomegalovirus (CMV) promoter on day 1. Expression of human apolipoprotein E3 (hApoE) in ApoE knockout mice provided a dose-dependent correction of their hypercholesterolemia. Liver-specific sustained transgene expression can be obtained at very high levels from optimized pDNA vectors, without the use of integration systems. Such vectors will further facilitate biological studies of genes in vivo and may find application in gene therapy.

Systemic AAV-9 transduction in mice is influenced by animal age but not by the route of administration

Adeno-associated virus (AAV) serotype-9 (AAV-9) has attracted great attention as an optimal vehicle for body-wide gene delivery. Here we examined the effect of animal age (newborn vs adult) and the route of administration (intravenous vs intra-arterial) on systemic AAV-9 transduction. We delivered an alkaline phosphatase (AP) reporter gene AAV vector (AV.RSV.AP) to either newborn (via either the facial vein or the left ventricular cavity) or adult (via tail vein) C57Bl/10 mice. At 12 weeks' postinfection, we examined the AP expression. We observed efficient transduction in multiple skeletal muscles and the heart, irrespective of the age or delivery route. However, the soleus muscle, which consists mainly of slow-twitch myofibers, was poorly transduced. Besides striated muscle, we also found consistent high-level transduction in the lung. Abundant AP-positive cells were seen in alveolar cells and vasculature, but not in bronchioles. Interestingly, several organs demonstrated an age-dependent profile. In particular, the aorta, liver and kidney were preferentially transduced in adult mice while the inner layer of retina was strongly transduced only following the neonatal administration. Taken together, our results demonstrate the robustness of intravascular AAV-9 delivery for muscle and lung gene therapy applications. The unique expression patterns in the aorta, liver, kidney and retina call for special attention when designing AAV-9 gene therapy applications for these organs.

Establishment of a non-invasive mouse reporter model for monitoring in vivo pdx-1 promoter activity

It is well known that pancreatic and duodenal homeobox gene-1 (PDX-1) plays a crucial role in β-cell differentiation, and maintaining mature β-cell function. Thus, it is important to understand how pdx-1 gene is regulated under various pathophysiological conditions in vivo. In this study, to non-invasively and quantitatively monitor pdx-1 promoter activity in vivo, we constructed a pdx-1 promoter-SEAP-IRES-GFP reporter plasmid. In this construct, the −4.6 kb pdx-1 promoter region sufficient for driving β-cell-selective PDX-1 expression was inserted to the upstream of the secreted alkaline phosphatase (SEAP) reporter gene. It is noted here that the pdx-1 promoter-mediated SEAP activity can be distinguished from endogenous alkaline phosphatase activity. First, we transfected the construct in mouse β-cell line MIN6 and human hepatocellular carcinoma cell line HepG2. SEAP activity was readily detected in the media of MIN6 cells, but not in HepG2 cells. These results indicate that this construct specifically reports β-cell-specific pdx-1 promoter activity in a cell culture system. Based on these in vitro findings, we next generated transgenic mice using the same construct. SEAP activity was readily detected in serum of the transgenic mice, but not in their littermate mice. Furthermore, SEAP activity was detected in protein extract from the transgenic pancreas and slightly from the transgenic duodenum, but not from the liver, and brain. These results indicate that serum SEAP activity likely represents in vivo pdx-1 promoter activity. This transgenic mouse model would be useful to non-invasively monitor in vivo pdx-1 promoter activity and to screen new molecules which regulate PDX-1 expression.

The effects of antidepressants on cyclic AMP-response element-driven gene transcription in a model cell system

The effects of the antidepressant drugs clomipramine (CLOM), desipramine (DMI), tianeptine (TIAN) and of norfluoxetine (NORF, the active metabolite of fluoxetine), were investigated in CHO cells expressing human β 2 adrenoceptors and a secreted placental alkaline phosphatase (SPAP) reporter gene to determine their actions on cyclic AMP-driven gene transcription. After 18 h of exposure, CLOM, DMI and NORF, but not TIAN, had biphasic effects on 1 μM isoprenaline-stimulated SPAP fsproduction with concentrations between 10 nM and 1 μM enhancing the maximal ( E max) SPAP response, without changing EC 50 values, but higher concentrations produced marked inhibitory effects. At nanomolar concentrations, CLOM and DMI increased expression of phospho-CREB (cyclic AMP response element binding protein). NORF was less effective but did significantly increase phospho-CREB at a concentration of 200 nM. TIAN had no effect. None of the antidepressants had any effect on CREB expression, nor on the accumulation of cyclic AMP. After prolonged exposure (7–21 days) to a low concentration (200 nM) of the antidepressants, the enhanced E max values for SPAP production evident after 18 h were not maintained but CLOM and DMI induced a significant leftward shift in the isoprenaline EC 50 after a 7-day period of treatment and this was sustained at the 21 day time point. TIAN did not produce any significant changes. The results demonstrate that, in vitro, some but not all antidepressants can modify gene transcription via monoamine and cyclic AMP-independent mechanisms. The in vivo adaptive responses to TIAN probably involve alterations in different gene sets to those affected by other antidepressants.

Muscle creatine kinase/SV40 hybrid promoter for muscle-targeted long-term transgene expression

Gene therapy for congenital protein deficiencies requires lifelong expression of a deficient protein. Current gene therapy approaches preferentially employ the strong cytomegalovirus (CMV) promoter/enhancer or its derivative CAG promoter; however, these promoters provide only temporary transgene expression. To create a promoter that enables long-lasting expression in muscle, hybrid promoters were constructed by coupling the muscle creatine kinase (MCK) enhancer to various strong promoters for enhancement of tissue specificity and improved transcriptional activity. A hybrid promoter containing the MCK enhancer and the simian virus 40 promoter (MCK/SV40 promoter) yielded long-term (>6 months) expression of a human secretory alkaline phosphatase (huSEAP) reporter gene following electrotransfer of the plasmid into mice, whereas expression using a conventional CMV or CAG promoter faded away within a few weeks. To explore the mechanism behind the sustained expression obtained with the MCK/SV40 promoter, mice were immunized with a LacZ expression plasmid driven by MCK/SV40 or a conventional promoter. Minimal cellular and humoral responses to LacZ were observed in MCK/SV40 promoter-treated animals, and mouse SEAP gene expression in vivo was successfully maintained by both the MCK/SV40 and conventional promoters. These results suggest that the lower immunogenicity of the MCK/SV40 promoter contributed to long-lasting gene expression in mice. Therefore, the MCK/SV40 promoter may provide the basis for development of an effective transgene expression cassette for treatment of congenital protein deficiencies in which therapeutic proteins are recognized as foreign by the host immune system.

The Chemopreventive Effect of Retinoids on Cellular NF-κB Activity Induced by NMU and NEU in Human Malignant Keratinocytes

Retinoids have been shown to be effective in suppressing tumor development when chemical carcinogens such as N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) were used to induce mammary tumors in a variety of animal models. However, the molecular mechanisms associated with the retinoid-mediated chemopreventive process, as linked to transcription factor NF-kappaB activation, for chemoprevention have not been elucidated. The purpose of this study was to determine the implications of NF-kappaB activation on the chemopreventive role of retinoids and their effect on cellular NF-kappaB activity that's induced by known alkylating chemical carcinogens such as NMU and NEU in human transfectant squamous cell carcinoma (SCC-13) cells. The activity of NF-kappaB, as regulated by chemical carcinogens and retinoids, was determined in cultured human SCC-13 keratinocytes that were transfected with the pNF-kappaB-SEAP-NPT plasmid; this permitted the expression of the secretory alkaline phosphatase (SEAP) reporter gene in response to the NF-kappaB activity, and the plasmid contained the neomycin phosphotransferase (NPT) gene, which confers resistance to geneticin. The reporter enzyme activity was measured using a fluorescence detection assay method. All-trans retinoic acid and 13-cis retinoic acid induced a reduction of NF-kappaB activity up to 64% and 65%, respectively, compared to the control. For the treatment of the human transfectant cells with chemical carcinogens, all-trans retinoic acid (5 mM) and 13-cis retinoic acid (5 mM) downregulated the cellular NF-kappaB activation up to 83% and 85% compared to the NF-kappaB activity that was upregulated by NMU (5microM) and NEU (5microM), respectively. These results suggest that the chemopreventive effect of retinoids may be mediated by the downregulated activation of NF-kappaB and that retinoids are implicated in the activation of NF-kappaB in human skin cells.

Analysis on the promoter region of human decidual prolactin gene in the progesterone-induced decidualization and cAMP-induced decidualization of human endometrial stromal cells

To elucidate the promoter region of human decidual prolactin (dPRL) gene in the human endometrial stromal cells (ESC). Various segments of the human dPRL promoter that direct the expression of the secreted alkaline phosphatase (SEAP) reporter gene were transfected into human ESC decidualized by estrogen (E) + progesterone (P) or cyclic AMP (cAMP) to identify E + P or cAMP responsive elements. The region between nucleotides -2038 and -1605 relative to the transcriptional initiation site includes two activator protein-1 (AP-1) sites, which both provided maximal response to E + P or cAMP in decidualized cells. When either AP-1 site was mutated, response in the promoter activity to both E + P or cAMP response showed a decrease compared with control. The region between -310 and -285 that contains consensus-binding sequences for transcription factors of CCAAT/Enhancer-binding proteins (C/EBP) contributed to E + P and cAMP response in decidualized cells. Also, the 5'-flanking region that extends 79 base pairs upstream, including an imperfect cAMP response element (CRE), contributed to E + P and cAMP response. In cells treated with E + P or cAMP for 10 days, mutant of C/EBP-binding site showed an increase in promoter activity comparing to dPRL-2038. In contrast, treatment with PKI showed a decrease in promoter activity in cells treated with E + P or cAMP alone. These results suggest that cAMP-induced region of the human dPRL promoter resides between -1862 and -1856, -1703 and -1697, -310 and -285, and that the sequences between -1862 and -1856, -1703 and -1697 of the promoter display E + P-induced promoter activity. Furthermore, the current study indicates that E + P or cAMP cooperatively regulate the dPRL gene transcription through some transcriptional factors such as C/EBP, CREB, and other cofactor(s), and that some repressor(s) or corepressor(s) may be involved in the C/EBP-binding site of the human dPRL promoter.

A Comparison of the Antagonist Affinities for the Gi- and Gs-Coupled States of the Human Adenosine A1-Receptor

The antagonist affinity for a given receptor is traditionally considered to be constant, reflecting the chemical nature of the specific ligand-receptor interaction. However, recent observations with all three beta-adrenoceptors have cast doubt on this basic pharmacological principle. The extent to which this finding applies to other G protein-coupled receptors and their interaction with different G proteins is unknown. Therefore, we studied the influence of different agonists on antagonist affinity measurements for Gi- and Gs-coupled conformations of the adenosine A1-receptor in Chinese hamster ovary cells stably expressing the human adenosine A1-receptor and a cAMP-response element (CRE)-secreted placental alkaline phosphatase reporter gene. Gi-coupled inhibition of [3H]cAMP accumulation via the A1-receptor was observed at low concentrations of agonist; however, a small increase in [3H]cAMP accumulation was also seen at higher agonist concentrations. This biphasic response was more evident for A1-stimulated CRE-gene transcription. The inhibitory component was abolished by pretreatment with pertussis toxin, whereas the stimulatory component was augmented, suggesting that the responses were due to an A1-Gi-coupled inhibition followed by an A1-Gs-coupled stimulation. However, the antagonist affinity values measured at the Gi-coupled and Gs-coupled conformations of the receptor were the same in both functional responses and whole-cell binding. Thus, in marked contrast to the beta-adrenoceptors, the A1-receptor conforms to the long-held principle of pharmacology that antagonist affinity measurements are constant regardless of the response being measured and the competing agonist used to stimulate that response. This was true even when the receptor was shown, in the same assay, to exist in two different conformational states coupled to two different G proteins.

Metal ions induced heat shock protein response by elevating superoxide anion level in HeLa cells transformed by HSE-SEAP reporter gene

The aim of this work is to define the relationship between heat shock protein (HSP) and reactive oxygen species (ROS) in the cells exposed to different concentrations of metal ions, and to evaluate a new method for tracing the dynamic levels of cellular reactive oxygen species using a HSE-SEAP reporter gene. The expression of heat shock protein was measured using a secreted alkaline phosphatase (SEAP) reporter gene transformed into HeLa cell strain, the levels of superoxide anion ( O 2 −) and hydrogen peroxide (H 2O 2) were determined by NBT reduction assay and DCFH staining flow cytometry (FCM), respectively. The experimental results demonstrated that the expression of heat shock protein induced by metal ions was linearly related to the cellular superoxide anion level before cytotoxic effects were observed, but not related to the cellular hydrogen peroxide level. The experimental results suggested that metal ions might induce heat shock protein by elevating cellular superoxide anion level, and thus the expression of heat shock protein indicated by the HSE-SEAP reporter gene can be an effective model for monitoring the dynamic level of superoxide anion and early metal-induced oxidative stress/cytotoxicity.

Lumbar Puncture Delivery of Bone Marrow Stromal Cells in Spinal Cord Contusion: A Novel Method for Minimally Invasive Cell Transplantation

Cell transplantation as a treatment for spinal cord injury is a promising therapeutic strategy whose effective clinical application would be facilitated by non-invasive delivery protocols. Cells derived from the bone marrow are particularly attractive because they can be obtained easily, expanded to large numbers and potentially used for autologous as well as allogeneic transplantation. In this study we tested the feasibility of a novel minimally invasive method--lumbar puncture (LP)--for transplanting bone marrow stromal stem cells (MSC) into a clinically relevant spinal cord contusion model. We further sought to determine optimal protocols for performing such minimally invasive cell transplantation. Sprague-Dawley rats received a moderate contusion injury at the midthoracic level followed by LP transplantation of MSC derived from transgenic rats that express the human placental alkaline phosphatase (AP) reporter gene. The recipients were analyzed histologically to evaluate the extent of cell delivery and survival at the injury site. We found that MSC delivered by LP reached the contused spinal cord tissues and exerted a significant beneficial effect by reducing cyst and injury size. Transplantation within 14 days of injury provided significantly greater grafting efficiency than more delayed delivery, and increasing MSC dosage improved cell engraftment. The techniques described here can easily be translated to patients, thus accelerating clinical application of stem cell therapies.

332. Routes of Ligand Administration That Give Efficient and Robust In Vivo Induction with the RheoSwitch® Therapeutic System

The RheoSwitch|[reg]| Therapeutic System (RTS) is used to regulate both the timing and level of gene expression in response to a systemically delivered small molecule ligand, Activator Drug. This inducible gene regulation system may have utility in human gene therapies that require a specifically regulated level of gene expression, where it is desirable to terminate the therapy when it is no longer needed, or where continuation of the therapy would be unsafe. The version of RTS used in these studies was comprised of an ecdysone receptor (EcR) fused to a GAL4 DNA binding domain, a chimeric retinoid X receptor fused to a VP16 activation domain (pVP16-Hs- LmRXR) , a human secreted alkaline phosphatase (hSEAP) reporter gene placed under the control of a 6xGAL4 response element, and a transthyretin minimal promoter (p6xGAL4-TTR-SEAP). The RTS was electroporated into the quadriceps of C57BL/6 mice and Activator Drug was administered by either intraperitoneal (IP) injection, intramuscular (IM) injection, subcutaneous injection, oral gavage, or administration in the rodent chow. Although ligand-dependent gene expression was observed by all five routes of administration, Activator Drug mixed in chow was easiest to administer, could be administered continuously and may mimic administration by capsule. As an example, six groups of mice (n = four/group) were administered various concentrations of Activator Drug in rodent chow 38 days after RTS electroporation. Activator Drug was given at a rate of 300, 150, 100, 50, 25, or 12.5 mg/kg chow for 21 days, corresponding to approximate total daily Activator Drug doses of 45, 23, 15, 8, 4 and 2 mg/kg/day, respectively. In this study, both the rate of ligand-mediated induction and the magnitude of ligand-mediated hSEAP expression were dependent on the concentration of ligand in the chow. Human SEAP reached a ligand- dose dependent plateau in mice receiving 100, 150, or 300 mg/kg chow two weeks after initiation of ligand administration. The threshold for induction was observed with 50 mg Activator Drug/kg chow, which showed a small induction of hSEAP that was slightly above background. In another long-term expression study, four groups of severe combined immunodeficient (SCID) mice (n = four/group) were administered Activator Drug in chow or by single IP injection at various times after electroporation. Activator Drug administration was started at 3, 32, or 76 days after RTS electroporation. In mice receiving Activator Drug by IP injection, gene expression could be cycled on and off by repeated IP administration. Likewise, gene expression could be similarly cycled when mice were put on and taken off chow containing Activator Drug. Gene expression remained |[ldquo]|on|[rdquo]| when mice were chronically maintained on chow containing Activator Drug. These studies establish that ligand-dependent induction of transgene expression can be obtained by various routes of ligand administration, and that gene expression can be sustained for long periods of time by chronic administration of ligand in chow. Further, the results demonstrate that transgene expression under control of RTS can be cycled on and off by repeat exposure to Activator Drug.

Krueppel-like transcription factor 11 (KLF11) inhibits activity of the human proinsulin promoter in pancreatic beta-cells

KLF11 (TIEG2) is a member of the Sp1/KLF transcription factor family which plays an important role in mammalian gene regulation. KLF11 is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here we investigated endogenous expression of KLF11 in pancreatic beta-cells and analyzed the effects of KLF11 overexpression on human proinsulin promoter activity. RT-PCR revealed endogenous KLF11 mRNA expression in whole rat pancreas, human pancreatic islets and rat INS-1 beta-cells. Glucose stimulation does not seem to modulate KLF11 expression in INS-1 beta-cells. Transient co-transfection of human proinsulin promoter driven secreted alkaline phosphatase (SEAP) reporter gene vectors with KLF11 expression plasmids in INS-1 and betaTC3 beta-cells demonstrates dose-dependent, but glucose-independent KLF11-mediated repression of insulin promoter activity. 5((prime))-deletion analysis of the human proinsulin promoter sequence and gel-shift assays (EMSA) of protein/DNA binding indicate specific GC and CACCC boxes within the human proinsulin promoter as potential KLF11 binding sites. Further, KLF11 may interfere with SP1 binding sites at these response elements. In summary, we have identified Krueppel-like transcription factor 11 (KLF11) as a novel negative transcriptional regulator of human proinsulin gene expression in pancreatic beta-cells. Further we have characterized specific KLF11 DNA-response elements within the human proinsulin promoter. Impairment of KLF11-dependent transcriptional regulation of proinsulin expression may contribute to beta-cell dysfunction seen in the pathogenesis of diabetes mellitus.

561. Novel Tissue-Specific Regulatory Cassettes Direct High-Level Transgene Expression in Skeletal and Cardiac Muscle

Top of pageAbstract Systemic delivery of rAAV6 vectors can achieve efficient transduction of the entire striated musculature, making this an attractive strategy for gene therapy of Duchenne Muscular Dystrophy. However, this delivery method also transduces cells in many non-muscle tissues, which may cause therapeutic problems including toxicity and a transgene-directed immune response. These problems could be avoided by limiting transgene expression through the use of muscle-specific promoters. Unfortunately, the expression levels of such promoters following systemic delivery of rAAV vectors are lower than viral promoters in most skeletal muscles and negligible in cardiac muscle. The goal of this project is to design regulatory cassettes that drive high levels of skeletal and cardiac muscle-specific transgene expression while also being sufficiently short (<800bp) to fit into rAAV vectors containing microdystrophin cDNA (3.8-kb). Our lab has developed a series of muscle-specific regulatory cassettes based on the enhancer and promoter of the murine muscle creatine kinase (MCK) gene. The CK6 cassette, previously thought to be the strongest modification, has higher activity than the wild type cassette in skeletal muscle, but very low activity in cardiac and diaphragm muscle. We have designed new cassettes to increase expression, and their activities have been evaluated in various muscles and non-muscle tissues following systemic delivery of rAAV6 vectors expressing a human placental alkaline phosphatase reporter gene. First, we designed a 570-bp MCK-based cassette, CK7, which exhibited activity levels similar to CK6 in quadriceps, a predominantly fast-twitch muscle, but had significantly higher activity in cardiac muscle, diaphragm, and soleus, a predominantly slow-twitch muscle. To further improve expression, especially in cardiac muscle, we added a 190-bp enhancer from the murine alpha-myosin heavy chain gene (|[alpha]|MHC) to the CK7 cassette to produce MHCK7, a 770-bp cassette that has the highest overall activity. When compared to CK6, the activity of MHCK7 was about 400-fold higher in cardiac muscle and 10-fold higher in soleus, which was equivalent to the activity of the CMV promoter. Staining for AP activity in cross-sections of both muscles revealed strong expression in virtually all fibers. The activity in diaphragm was about 40-fold higher, but was still considerably lower than the CMV promoter, with only about 20% of the fibers staining positive. Finally, the activity in quadriceps muscle was equivalent to CK6. The activity of MHCK7 in kidney, aorta, brain, testes, and intestine was at background levels, while a very slight increase in activity, about 50|[ndash]|200-fold lower than in muscle, was detected in liver, spleen, and lung. Based on these results, the MHCK7 cassette is currently being tested with respect to its capacity for driving microdystrophin expression in mdx mice following intramuscular and systemic injections of rAAV6 vectors.