Induction Of GFP Expression Research Articles

Agrochemical control of gene expression using evolved split RNA polymerase. II.

Agrochemical inducible gene expression system provides cost-effective and orthogonal control of energy and information flow in bacterial cells. However, the previous version of Mandipropamid inducible gene expression system (Mandi-T7) became constitutively active at room temperature. We moved the split site of the eRNAP from position LYS179 to position ILE109. This new eRNAP showed proximity dependence at 23 °C, but not at 37 °C. We built Mandi-T7-v2 system based on the new eRNAP and it worked in both Escherichia coli and Agrobacterium tumefaciens. We also induced GFP expression in Agrobacterium cells in a semi-in vivo system. The modified eRNAP when combined with the leucine zipper-based dimerization system, behaved as a cold inducible gene expression system. Our new system provides a means to broaden the application of agrochemicals for both research and agricultural application. Portions of this text were previously published as part of a preprint (https://www.biorxiv.org/content/10.1101/2024.04.02.587689v1).

RNA Aptamer-Mediated Gene Activation Systems for Inducible Transgene Expression in Animal Cells.

RNA expression analyses can be used to obtain various information from inside cells, such as physical conditions, the chemical environment, and endogenous signals. For detecting RNA, the system regulating intracellular gene expression has the potential for monitoring RNA expression levels in real time within living cells. Synthetic biology provides powerful tools for detecting and analyzing RNA inside cells. Here, we devised an RNA aptamer-mediated gene activation system, RAMGA, to induce RNA-triggered gene expression activation by employing an inducible complex formation strategy grounded in synthetic biology. This methodology connects DNA-binding domains and transactivators through target RNA using RNA-binding domains, including phage coat proteins. MS2 bacteriophage coat protein fused with a transcriptional activator and PP7 bacteriophage coat protein fused with the tetracycline repressor (tetR) can be bridged by target RNA encoding MS2 and PP7 stem-loops, resulting in transcriptional activation. We generated recombinant CHO cells containing an inducible GFP expression module governed by a minimal promoter with a tetR-responsive element. Cells carrying the trigger RNA exhibited robust reporter gene expression, whereas cells lacking it exhibited no expression. GFP expression was upregulated over 200-fold compared with that in cells without a target RNA expression vector. Moreover, this system can detect the expression of mRNA tagged with aptamer tags and modulate reporter gene expression based on the target mRNA level without affecting the expression of the original mRNA-encoding gene. The RNA-triggered gene expression systems developed in this study have potential as a new platform for establishing gene circuits, evaluating endogenous gene expression, and developing novel RNA detectors.

Bisphenol A analogues induce a feed-forward estrogenic response in zebrafish

Because exposure to bisphenol A (BPA) has been linked to health problems in humans and wildlife, BPA analogues have been synthesized to be considered as replacement molecules. We here have examined estrogenic activity of BPA and five of its analogues, BPAF, BPE, BPC, BPC-Cl, and BPS by a combination of zebrafish-based in vivo and in vitro assays. We used transgenic estrogen reporter (5xERE:GFP) fish to study agonistic effects of bisphenols. Exposures to BPA, BPAF, BPE, and BPC, induced GFP expression in estrogen reporter fish at low exposure concentrations in the heart valves and at higher concentrations in the liver, whereas BPC-Cl activated GFP expression mainly in the liver, and BPS faintly in the heart only. The in vivo response was compared to in vitro estrogenicity of bisphenol exposure using reporter cells that express the zebrafish estrogen receptors driving expression of an estrogen response element (ERE)-luciferase reporter. In these cells, BPA, BPAF, BPC, BPE and BPS preferentially activated Esr1, whereas BPC-Cl preferentially activated Esr2a. By quantitative PCR we found that exposure to BPAF induced expression of the classical estrogen target genes vtg1, esr1, and cyp19a1b in a concentration response manner, but the most responsive target gene was f13a1a. Exposure to BPC-Cl resulted in a different expression pattern of vtg1 and f13a1a with an activation at low concentrations, followed by a declining expression at higher concentrations. Because expression of f13a1a was strongly activated by all compounds tested, we suggest including this mRNA as a biomarker for estrogenicity in larval fish. We further showed that exposure to BPAF and BPC-Cl increased E2 levels in zebrafish larvae, indicating that bisphenol exposures result in a feed-forward response that can further augment the estrogenic activity of these compounds.

Generation of a recombinant version of a biologically active cell-permeant human HAND2 transcription factor from E. coli

Transcription factor HAND2 has a significant role in vascularization, angiogenesis, and cardiac neural crest development. It is one of the key cardiac factors crucial for the enhanced derivation of functional and mature myocytes from non-myocyte cells. Here, we report the generation of the recombinant human HAND2 fusion protein from the heterologous system. First, we cloned the full-length human HAND2 gene (only protein-coding sequence) after codon optimization along with the fusion tags (for cell penetration, nuclear translocation, and affinity purification) into the expression vector. We then transformed and expressed it in Escherichia coli strain, BL21(DE3). Next, the effect (in terms of expression) of tagging fusion tags with this recombinant protein at two different terminals was also investigated. Using affinity chromatography, we established the one-step homogeneous purification of recombinant human HAND2 fusion protein; and through circular dichroism spectroscopy, we established that this purified protein had retained its secondary structure. We then showed that this purified human protein could transduce the human cells and translocate to its nucleus. The generated recombinant HAND2 fusion protein showed angiogenic potential in the ex vivo chicken embryo model. Following transduction in MEF2C overexpressing cardiomyoblast cells, this purified recombinant protein synergistically activated the α-MHC promoter and induced GFP expression in the α-MHC-eGFP reporter assay. Prospectively, the purified bioactive recombinant HAND2 protein can potentially be a safe and effective molecular tool in the direct cardiac reprogramming process and other biological applications.

Agrochemical control of gene expression using evolved split RNA polymerase.

Chemically-inducible gene expression systems are valuable tools for rational control of gene expression both for basic research and biotechnology. However, most chemical inducers are confined to certain groups of organisms. Therefore, dissecting interactions between different organisms could be challenging using existing chemically-inducible systems. We engineered a mandipropamid-induced gene expression system (Mandi-T7) based on evolved split T7 RNAP system. As a proof-of-principle, we induced GFP expression in E. coli cells grown inside plant tissue.

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System.

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Possible application of muscle specific conditional mouse-derived induced pluripotent stem cells for muscle research

The Cre-driver mouse line, which allows for in vivo regulation of target gene(s) in specific cells, is an indispensable tool for recent muscle research. In this study, I aimed to explore new applications of muscle specific Cre-driver mouse line in muscle research. For this purpose, I generated an iPS cells from a myofiber specific conditional mouse with tamoxifen inducible GFP expression, and then I checked whether homologous recombination was induced in the iPS-derived myogenic cells by tamoxifen administration. Fibroblasts were isolated from the tails of Myf6CE/wt::CAG-EGFP mice, which expressed GFP specifically in Myf6 lineages by tamoxifen injection, and then iPS cells was generated by transfection with a vector based on sendai-virus and containing OSKM genes. Muscle specific conditional mouse-derived iPS cells (mCM-iPSCs) were successfully differentiated to myogenic cells, such as Pax7+ muscle progenitors, MyoD+ myoblasts, and MHC+ myotubes, under myogenic differentiation conditions. Using this model, I examined whether homologous recombination was induced in mCM-iPSC-derived myotubes by 4-hydroxytamoxifen (4OH-TAM) administration. As a result, multinucleated myotubes showed GFP expression, while no GFP signals were detected in both Pax7+ muscle progenitor and non-myogenic cells. These results indicated that homologous recombination could be induced in mCM-iPSC–derived myotubes by tamoxifen administration, and that this system operated normally even in reprogrammed cells. Also, I evidenced that GFP reporter was expressed in myoblasts in addition to multinucleated myotubes when tamoxifen-pulse was applied at an early phase of myogenesis. Taken together, Myf6CE/wt::CAG-EGFP mouse-derived iPS cells reproduced at least in part Myf6 expression during mouse myogenesis. This study demonstrated a novel application of muscle specific conditional mouse in addition to in vivo application, and mCM-iPSCs could also be used in in vitro investigations with muscle specific conditional knock-out mouse.

Wnt signaling regulates proliferation and differentiation of radial glia in regenerative processes after stab injury in the optic tectum of adult zebrafish.

Zebrafish have superior abilities to generate new neurons in the adult brain and to regenerate brain tissue after brain injury compared with mammals. There exist two types of neural stem cells (NSCs): neuroepithelial-like stem cells (NE) and radial glia (RG) in the optic tectum. We established an optic tectum stab injury model to analyze the function of NSCs in the regenerative condition and confirmed that the injury induced the proliferation of RG, but not NE and that the proliferated RG differentiated into new neurons after the injury. We then analyzed the involvement of Wnt signaling after the injury, using a Wnt reporter line in which canonical Wnt signaling activation induced GFP expression and confirmed that GFP expression was induced specifically in RG after the injury. We also analyzed the expression level of genes related to Wnt signaling, and confirmed that endogenous Wnt antagonist dkk1b expression was significantly decreased after the injury. We observed that Wnt signal inhibitor IWR1 treatment suppressed the proliferation and differentiation of RG after the injury, suggesting that up-regulation of Wnt signaling in RG after the stab injury was required for optic tectum regeneration. We also confirmed that Wnt activation by treatment with GSK3β inhibitor BIO in uninjured zebrafish induced proliferation of RG in the optic tectum. This optic tectum stab injury model is useful for the study of the molecular mechanisms of brain regeneration and analysis of the RG functions in physiological and regenerative conditions.

Intestinal Stem Cell Pool Regulation in Drosophila.

SummaryIntestinal epithelial renewal is mediated by intestinal stem cells (ISCs) that exist in a state of neutral drift, wherein individual ISC lineages are regularly lost and born but ISC numbers remain constant. To test whether an active mechanism maintains stem cell pools in the Drosophila midgut, we performed partial ISC depletion. In contrast to the mouse intestine, Drosophila ISCs failed to repopulate the gut after partial depletion. Even when the midgut was challenged to regenerate by infection, ISCs retained normal proportions of asymmetric division and ISC pools did not increase. We discovered, however, that the loss of differentiated midgut enterocytes (ECs) slows when ISC division is suppressed and accelerates when ISC division increases. This plasticity in rates of EC turnover appears to facilitate epithelial homeostasis even after stem cell pools are compromised. Our study identifies unique behaviors of Drosophila midgut cells that maintain epithelial homeostasis.

Recombinant adeno-associated virus serotype 9 in a mouse model of atherosclerosis: Determination of the optimal expression time invivo.

Adeno-associated virus 9 (AAV9) has been identified as one of the optimal gene transduction carriers for gene therapy. The aim of the present study was to determine the gene transfection efficiency and safety of an AAV9 vector produced using a recombinant baculovirus (rBac)-based system. AAV9-cytomegalovirus (CMV)-green fluorescent protein was produced using an rBac system and the resulting vector particles were injected intravenously into mice. Animals were sacrificed at 14, 21, 28, 35, 60, 90 and 120 days following injection. GFP expression in aortic vasculature and aortic plaques in C57/6B and apolipoprotein E−/− mice was analyzed by fluorescence imaging and western blotting. In vivo analyses of biological markers of liver and heart damage, and renal function, as well as in vitro terminal deoxynucleotidyl transferase dUTP nick end labeling analysis were used to determine the toxicity of the AAV9 carrier. The findings of the present study demonstrated that AAV9 viral vectors packaged using the rBac system functioned appropriately in arteriosclerosis plaques. The CMV promoter significantly induced GFP expression in the vascular plaque in a time-dependent manner. AAV9-CMV viral particles did not lead to heart, liver or renal damage and no change in apoptotic rate was identified. These findings indicated that AAV9-CMV may be effectively and safely used to transfect genes into atherosclerotic plaques.

In situ real-time evaluation of radiation-responsive promoters in the extremely radioresistant microbe Deinococcus radiodurans.

A third generation promoter probe shuttle vector pKG was constructed, using the green fluorescent protein as a reporter, for in situ evaluation of Deinococcal promoter activity in Escherichia coli or Deinococcus radiodurans. The construct yielded zero background fluorescence in both the organisms, in the absence of promoter sequences. Fifteen Deinococcal promoters, either harbouring Radiation and Desiccation Response Motif (RDRM) or not, were cloned in vector pKG. Only the RDRM-promoter constructs displayed (i) gamma radiation inducible GFP expression in D. radiodurans, following gamma irradiation, (ii) DdrO-mediated repression of GFP expression in heterologous E. coli, or (iii) abolition in GFP induction following gamma irradiation, in pprI mutant of D. radiodurans. Utility of pKG vector for real-time in situ assessment of Deinococcal promoter function was, thus, successfully demonstrated.

491. Messenger RNA (mRNA)-Based Gene Therapy for Introducing Anti-Apoptotic Factor

In gene therapy, mRNA delivery lacks the risk of random integration to host genome in contrast to DNA delivery. In introduction of anti-apoptotic factors, uncontrollably enhanced and prolonged expression of such factors after genomic integration would cause cancer, and thus DNA delivery cannot be used for introducing these factors in clinical settings. In this study, we tried to treat fulminant hepatitis, a disease associated with excessive apoptosis, by mRNA-based introduction of an anti-apoptotic factor, Bcl-2. For efficient mRNA delivery with minimal inflammatory responses, mRNA was loaded into our original carrier, polyplex nanomicelles (Adv Drug Deliv Rev 2001, 47, 113, PLoS One 2013, 8, e56220), which are composed of polyethyleneglycol-coated surface and mRNA-containing core, for hydrodynamic injection into mouse liver.In a reporter assay using GFP-expressing mRNA, the nanomicelles effectively introduced mRNA into liver cells by preventing degradation of mRNA, resulting in significantly higher level of GFP expression in liver compared to that after naked mRNA injection. This result is quite different from that of naked plasmid DNA (pDNA) delivery, strongly suggesting that mRNA is unstable in mouse body, and a proper carrier is required for mRNA delivery.In tissue distribution analysis of GFP expression, mRNA delivery induced GFP expression uniformly in almost 100% of liver cells, whereas only a small percentage of liver cells became GFP positive after pDNA delivery. After delivery of Cy5-labeled pDNA, nuclear localization of pDNA was undetected in a large percentage of cells, indicating that nuclear entry was a large obstacle in DNA delivery. In contrast, protein expression from mRNA requires only cytoplasmic uptake of mRNA, and this was observed in almost all liver cells, resulting in uniform GFP expression profile.Then, we performed the treatment of fulminant hepatitis using Bcl-2-expressing gene, after preparing a mouse model of the disease by injecting Fas ligand. After mRNA-based introduction of Bcl-2, the number of apoptotic cells in liver was significantly reduced when compared to saline-treated control. In contrast, delivery of Bcl-2-expressing pDNA had almost no therapeutic effect. It is suggested that, in pDNA delivery, a large percentage of cells were unprotected from apoptotic signals because of inhomogeneous protein expression profile. Thus, these results indicate that mRNA delivery is more suitable for introducing intracellular signaling factors than DNA delivery, because such factors require homogeneous expression to obtain therapeutic effect. This is in sharp contrast to the case of secretable factors, where efficient introduction to only a small percentage of cells is sufficient to obtain therapeutic effect.In summary, mRNA delivery has two advantages over pDNA delivery: homogeneous distribution profile of protein expression, and lack of random genomic integration. Thus, mRNA delivery is promising for the treatment of various intractable diseases associated with massive apoptosis.

Coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy

Retinal gene therapy could potentially affect the lives of millions of people suffering from blinding disorders. Yet, one of the major hurdles remains the delivery of therapeutic nucleic acids to the retinal target cells. Due to the different barriers that need to be overcome in case of topical or systemic administration, intravitreal injection is an attractive alternative administration route for large macromolecular therapeutics. Here it is essential that the therapeutics do not aggregate and remain mobile in the vitreous humor in order to reach the retina. In this study, we have evaluated the use of hyaluronic acid (HA) as an electrostatic coating for nonviral polymeric gene nanomedicines, p(CBA-ABOL)/pDNA complexes, to provide them with an anionic hydrophilic surface for improved intravitreal mobility. Uncoated polyplexes had a Z-averaged diameter of 108nm and a zeta potential of +29mV. We evaluated polyplexes coated with HA of different molecular weights (22kDa, 137kDa and 2700kDa) in terms of size, surface charge and complexation efficiency and noticed their zeta potentials became anionic at 4-fold molar excess of HA-monomers compared to cationic monomers, resulting in submicron ternary polyplexes. Next, we used a previously optimized ex vivo model based on excised bovine eyes and fluorescence single particle tracking (fSPT) microscopy to evaluate mobility in intact vitreous humor. It was confirmed that HA-coated polyplexes had good mobility in bovine vitreous humor, similar to polyplexes functionalized with polyethylene glycol (PEG), except for those coated with high molecular weight HA (2700kDa). However, contrary to PEGylated polyplexes, HA-coated polyplexes were efficiently taken up in vitro in ARPE-19 cells, despite their negative charge, indicating uptake via CD44-receptor mediated endocytosis. Furthermore, the HA-polyplexes were able to induce GFP expression in this in vitro cell line without apparent cytotoxicity, where coating with low molecular weight HA (22kDa) was shown to induce the highest expression. Taken together our experiments show that HA-coating of nonviral gene complexes is an interesting approach towards retinal gene therapy by intravitreal administration. To our knowledge, this is the first time electrostatic HA-coating of polyplexes with different molecular weights has been evaluated in terms of their suitability for intravitreal delivery of therapeutic nucleic acids towards the retina.

Antigen-dependent versus -independent activation of invariant NKT cells during infection.

CD1d-reactive invariant NKT cells (iNKT) play a vital role in determining the characteristics of immune responses to infectious agents. Previous reports suggest that iNKT cell activation during infection can be: 1) solely driven by cytokines from innate immune cells, 2) require microbial Ag, or 3) require self-Ag. In this study, we examined the role of Ag receptor stimulation in iNKT cells during several bacterial and viral infections. To test for Ag receptor signaling, Nur77(gfp) BAC transgenic mice, which upregulate GFP in response to Ag receptor but not inflammatory signals, were analyzed. iNKT cells in the reporter mice infected with mouse CMV produced IFN-γ but did not upregulate GFP, consistent with their reported CD1d-independent activation. However, two bacteria known to produce lipid Ags for iNKT cells induced GFP expression and cytokine production. In contrast, although Salmonella typhimurium was proposed to induce the presentation of a self-lipid, iNKT cells produced IFN-γ but did not upregulate GFP postinfection in vivo. Even in CD1d-deficient hosts, iNKT cells were still able to produce IFN-γ after S. typhimurium infection. Furthermore, although it has been proposed that endogenous lipid presentation is a result of TLR stimulation of APCs, injection of different TLR agonists led to iNKT cell IFN-γ but not increased GFP expression. These data indicate that robust iNKT cell responses to bacteria, as well as viruses, can be obtained in the absence of antigenic stimulation.

Hierarchical Functional Specificity of Cytosolic Heat Shock Protein 70 (Hsp70) Nucleotide Exchange Factors in Yeast

Heat shock protein 70 (Hsp70) molecular chaperones play critical roles in protein homeostasis. In the budding yeast Saccharomyces cerevisiae, cytosolic Hsp70 interacts with up to three types of nucleotide exchange factors (NEFs) homologous to human counterparts: Sse1/Sse2 (Heat shock protein 110 (Hsp110)), Fes1 (HspBP1), and Snl1 (Bag-1). All three NEFs stimulate ADP release; however, it is unclear why multiple distinct families have been maintained throughout eukaryotic evolution. In this study we investigate NEF roles in Hsp70 cell biology using an isogenic combinatorial collection of NEF deletion mutants. Utilizing well characterized model substrates, we find that Sse1 participates in most Hsp70-mediated processes and is of particular importance in protein biogenesis and degradation, whereas Fes1 contributes to a minimal extent. Surprisingly, disaggregation and resolubilization of thermally denatured firefly luciferase occurred independently of NEF activity. Simultaneous deletion of SSE1 and FES1 resulted in constitutive activation of heat shock protein expression mediated by the transcription factor Hsf1, suggesting that these two factors are important for modulating stress response. Fes1 was found to interact in vivo preferentially with the Ssa family of cytosolic Hsp70 and not the co-translational Ssb homolog, consistent with the lack of cold sensitivity and protein biogenesis phenotypes for fes1Δ cells. No significant consequence could be attributed to deletion of the minor Hsp110 SSE2 or the Bag homolog SNL1. Together, these lines of investigation provide a comparative analysis of NEF function in yeast that implies Hsp110 is the principal NEF for cytosolic Hsp70, making it an ideal candidate for therapeutic intervention in human protein folding disorders.

Conditional Gene Expression in Chlamydia trachomatis Using the Tet System

Chlamydia trachomatis is maintained through a complex bi-phasic developmental cycle that incorporates numerous processes that are poorly understood. This is reflective of the previous paucity of genetic tools available. The recent advent of a method for transforming Chlamydia has enabled the development of essential molecular tools to better study these medically important bacteria. Critical for the study of Chlamydia biology and pathogenesis, is a system for tightly controlled inducible gene expression. To accomplish this, a new shuttle vector was generated with gene expression controlled by the Tetracycline repressor and anhydryotetracycline. Evaluation of GFP expression by this system demonstrated tightly controlled gene regulation with rapid protein expression upon induction and restoration of transcription repression following inducer removal. Additionally, induction of expression could be detected relatively early during the developmental cycle and concomitant with conversion into the metabolically active form of Chlamydia. Uniform and strong GFP induction was observed during middle stages of the developmental cycle. Interestingly, variable induced GFP expression by individual organisms within shared inclusions during later stages of development suggesting metabolic diversity is affecting induction and/or expression. These observations support the strong potential of this molecular tool to enable numerous experimental analyses for a better understanding of the biology and pathogenesis of Chlamydia.

Human CD300C Delivers an Fc Receptor-γ-dependent Activating Signal in Mast Cells and Monocytes and Differs from CD300A in Ligand Recognition

CD300C is highly homologous with an inhibitory receptor CD300A in an immunoglobulin-like domain among the human CD300 family of paired immune receptors. To clarify the precise expression and function of CD300C, we generated antibodies discriminating between CD300A and CD300C, which recognized a unique epitope involving amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Notably, CD300C was highly expressed in human monocytes and mast cells. Cross-linking of CD300C by its specific antibody caused cytokine/chemokine production of human monocytes and mast cells. Fc receptor γ was indispensable for both efficient surface expression and activating functions of CD300C. To identify a ligand for CD300A or CD300C, we used reporter cell lines expressing a chimera receptor harboring extracellular CD300A or CD300C and intracellular CD3ζ, in which its unknown ligand induced GFP expression. Our results indicated that phosphatidylethanolamine (PE) among the lipids tested and apoptotic cells were possible ligands for both CD300C and CD300A. PE and apoptotic cells more strongly induced GFP expression in the reporter cells through binding to extracellular CD300A as compared with CD300C. Differential recognition of PE by extracellular CD300A and CD300C depended on different amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Interestingly, GFP expression induced by extracellular CD300C-PE binding in the reporter cells was dampened by co-expression of full-length CD300A, indicating the predominance of CD300A over CD300C in PE recognition/signaling. PE consistently failed to stimulate cytokine production in monocytes expressing CD300C with CD300A. In conclusion, specific engagement of CD300C led to Fc receptor γ-dependent activation of mast cells and monocytes.

Mechanical input restrains PPARγ2 expression and action to preserve mesenchymal stem cell multipotentiality

Exercise-generated signals are pro-osteogenic and anti-adipogenic within the marrow. In vitro studies indicate that mechanical signals directly block adipogenic differentiation through activation of β-catenin and by limiting PPARγ2 expression. Whether mechanically generated β-catenin can inhibit adipogenesis during PPARγ transactivation is unknown. We evaluated the ability of mechanical signals to limit adipogenesis in marrow derived mesenchymal stem cells (mdMSC) distal to activation of PPARγ. First, we established that mdMSC attained an adipogenic phenotype within 2–4days in the presence of rosiglitazone (1–25μM) and that β-catenin activation via GSK3β inhibition interfered with this process. Similarly, mechanical strain (3600cycles, 2% strain daily) inhibited adipogenesis at 3days, preventing rosiglitazone-induced PPARγ upregulation as well as aP2 and adiponectin protein expression. To assess whether a reduction in PPARγ expression was necessary for anti-adipogenic action, PPARγ2 was overexpressed: both mechanical strain and GSK3β inhibition prevented expression of aP2 and adiponectin proteins despite abundant PPARγ2 and its ligand. To understand the fate of single cells experiencing mechanical strain we generated mdMSC from aP2-GFP reporter expressing mice. Rosiglitazone treatment for 3days induced GFP expression in more than 80% of cells. Sorting by GFP expression revealed that the highest 20% of aP2-GFP expressing cells was responsible for the majority of adipogenic protein expression. This highly expressing GFP fraction had a reduced ability to respond to an osteogenic stimulus: BMP-2 treatment increased osterix by 12-fold in contrast to the 42-fold increase in osterix expression that resulted from BMP-2 treatment of the bottom 75% of GFP expressing cells. This suggested that highly expressing aP2-GFP cells represented more terminally differentiated adipocytes, with reduced multipotentiality. Application of mechanical strain to aP2-GFP mdMSC treated with rosiglitazone caused a two-fold decrease in the size of the upper cell fraction, suggesting that mechanical strain preserved MSC in a multipotent state. Our data show that mechanical strain restricts adipogenesis both by limiting PPARγ2 expression and by preventing PPARγ action, protecting the potential of MSC to enter other lineages.

P106 Non-redundant in vivo functions of IFN-β protect against virus infection within the central nervous system

Introduction Previous studies showed that type I interferon receptor (IFNAR) triggering is critically required to control many different virus infections. In mice one IFN- β and 14 different IFN- α isotypes interact with IFNAR. Infection experiments with peripherally challenged IFN- β −/− mice did not reveal any overt phenotype suggesting that IFN- α and IFN- β played a redundant role under such conditions. Here we readdressed IFN- β function under conditions of intranasal (i.n.) virus infection. Methods IFN- β −/− and C57BL/6 mice were i.n. and i.v. infected with vesicular stomatitis virus (VSV) and their survival monitored. Serum IFN titers were determined by an ELISA method. Additionally reporter mice for the scrutiny of IFN- β expression (IFN- β Δ βluc / WT ) and IFNAR signaling (ISRE-eGFP) were used for in vivo and ex vivo analysis. Results Upon i.n. VSV infection IFN- β −/− mice showed a significantly enhanced vulnerability to lethal VSV infection. A previous study revealed that upon i.n. VSV instillation of C57BL/6 mice the virus readily infected olfactory sensory neurons and moved along axons into the glomerular layer of the olfactory bulb, where it was arrested in an IFNAR-dependent manner. After i.v. as well as i.n. VSV infection IFN- α , but no IFN- β , was detected in the serum. Analysis of IFN- β reporter mice revealed that within the central nervous system (CNS) protective IFN- β was induced primarily in the olfactory bulb, presumably by neurons. Interestingly, locally produced IFN- β was active also in distal brain areas as evidenced by induction of GFP expression in the cerebrum of i.n. infected ISRE-eGFP reporter mice. Of note ISRE-eGFP mice intercrossed with IFN- β −/− mice did not show local IFNAR signaling within the CNS. Conclusion In conclusion our experiments are in accordance with the model that upon virus infection of the CNS IFN- β responses are selectively induced within the olfactory bulb that stimulate also distal brain areas, whereas IFN- α s are primarily induced in the periphery and there account for protection of peripheral tissues.

Abstract 17172: Adiponectin Promoter Elements Combined with Liver-Specific microRNA-122 Target Sequences Specifically Target Adipose Tissue for Gene Transfer with Intravenous Delivery an AAV8 Vector

Objective: To develop an in vivo , systemic method of gene transfer specifically targeting adipose tissue. We assessed the hypothesis that systemic adeno-associated virus (AAV)-mediated vector delivery can transfer genes specifically to adipose tissue. Background: Obesity is a major risk factor for developing coronary heart disease. Adipose is an endocrine organ critical to energy intake and expenditure. Available data on adipose is limited, in part, because of the difficulties in adapting experimental techniques to a tissue with high lipid content. Few studies have investigated in vivo gene transfer in adipose tissue, and these studies used subcutaneous injections in obese mice. Method and Results: Tail vein IV injections of a luciferase expression vector in healthy C57BL/6 mice revealed that AAV serotype 8 (AAV8) best targeted adipose tissue. Tail vein injections of AAV8 were used to administer all subsequent vectors in five mice per vector. Mice were sacrificed 2-3 weeks after injection, and tissues were analyzed for transgene expression. Systemic delivery of a vector containing the GFP gene under control of a CMV promoter induced GFP transgene expression in most tissues tested, including subcutaneous, omental, gonadal, and brown fat depots. To restrict GFP expression to adipose depots, the CMV promoter was replaced with an adiponectin enhancer/promoter to make Adipo-GFP AAV. Systemic delivery of Adipo-GFP AAV induced GFP expression in all adipose depots tested. GFP expression in non-adipose tissues was negligible except for liver. To ameliorate unwanted liver expression, target sites for liver specific microRNA -122 were incorporated into the vector to make Adipo-GFP-miR122 AAV. Mice injected with Adipo-GFP-miR122 AAV no longer expressed GFP in liver, yet they expressed GFP in adipose tissue. Furthermore, mature adipocytes isolated from adipose tissue express GFP, indicating Adipo-GFP-miR122 vector targets mature adipocytes for transgene expression. Conclusion: AAV8-mediated systemic delivery of a specifically designed adipose expression vector can selectively target adipose tissue for in vivo gene transfer. This novel approach has therapeutic potential in obesity and downstream complications like insulin resistance and diabetes.