A high sensitivity ZENK monoclonal antibody to map neuronal activity in Aves

The transcription factor ZENK is an immediate early gene that has been employed as a surrogate marker to map neuronal activity in the brain. It has been used in a wide variety of species, however, commercially available antibodies have limited immunoreactivity in birds. To address this issue we generated a new mouse monoclonal antibody, 7B7-A3, raised against ZENK from the rock pigeon (Columba livia). We show that 7B7-A3 labels clZENK in both immunoblots and histological stainings with high sensitivity and selectivity for its target. Using a sound stimulation paradigm we demonstrate that 7B7-A3 can detect activity-dependent ZENK expression at key stations of the central auditory pathway of the pigeon. Finally, we compare staining efficiency across three avian species and confirm that 7B7-A3 is compatible with immunohistochemical detection of ZENK in the rock pigeon, zebra finch, and domestic chicken. Taken together, 7B7-A3 represents a useful tool for the avian neuroscience community to map functional activity in the brain

The expression, localisation and interactome of pigeon CRY2

Cryptochromes (CRY) are highly conserved signalling molecules that regulate circadian rhythms and are candidate radical pair based magnetoreceptors. Birds have at least four cryptochromes (CRY1a, CRY1b, CRY2, and CRY4), but few studies have interrogated their function. Here we investigate the expression, localisation and interactome of clCRY2 in the pigeon retina. We report that clCRY2 has two distinct transcript variants, clCRY2a, and a previously unreported splice isoform, clCRY2b which is larger in size. We show that clCRY2a mRNA is expressed in all retinal layers and clCRY2b is enriched in the inner and outer nuclear layer. To define the localisation and interaction network of clCRY2 we generated and validated a monoclonal antibody that detects both clCRY2 isoforms. Immunohistochemical studies revealed that clCRY2a/b is present in all retinal layers and is enriched in the outer limiting membrane and outer plexiform layer. Proteomic analysis showed clCRY2a/b interacts with typical circadian molecules (PER2, CLOCK, ARTNL), cell junction proteins (CTNNA1, CTNNA2) and components associated with the microtubule motor dynein (DYNC1LI2, DCTN1, DCTN2, DCTN3) within the retina. Collectively these data show that clCRY2 is a component of the avian circadian clock and unexpectedly associates with the microtubule cytoskeleton

pEPito: a significantly improved non-viral episomal expression vector for mammalian cells

<p>Abstract</p> <p>Background</p> <p>The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed <it>Cytomegalovirus </it>immediate early promoter (CMV-IEP) and directed into a 2000 bp long <it>matrix attachment region sequence </it>(MARS) derived from the human β-interferon gene. The original pEPI-1 vector contains two mammalian transcription units and a total of 305 CpG islands, which are located predominantly within the vector elements necessary for bacterial propagation and known to be counterproductive for persistent long-term transgene expression.</p> <p>Results</p> <p>Here, we report the development of a novel vector pEPito, which is derived from the pEPI-1 plasmid replicon but has considerably improved efficacy both <it>in vitro </it>and <it>in vivo</it>. The pEPito vector is significantly reduced in size, contains only one transcription unit and 60% less CpG motives in comparison to pEPI-1. It exhibits major advantages compared to the original pEPI-1 plasmid, including higher transgene expression levels and increased colony-forming efficiencies <it>in vitro</it>, as well as more persistent transgene expression profiles <it>in vivo</it>. The performance of pEPito-based vectors was further improved by replacing the CMV-IEP with the <it>human CMV enhancer/human elongation factor 1 alpha promoter </it>(hCMV/EF1P) element that is known to be less affected by epigenetic silencing events.</p> <p>Conclusions</p> <p>The novel vector pEPito can be considered suitable as an improved vector for biotechnological applications <it>in vitro </it>and for non-viral gene delivery <it>in vivo</it>.</p

Cadmium Telluride Quantum Dots as a Fluorescence Marker for Adipose Tissue Grafts

Plastic and reconstructive surgeons increasingly apply adipose tissue grafting in a clinical setting, although the anticipation of graft survival is insecure. There are only few tools for tracking transplanted fat grafts in vivo. Murine adipose tissue clusters were incubated with negatively charged, mercaptoproprionic acid-coated cadmium telluride quantumdots (QDs) emitting in the dark red or near infrared. The intracellular localization of QDs was studied by confocal laser scanning microscopy. As a result, the adipose tissue clusters showed a proportional increase in fluorescence with increasing concentrations (1, 10, 16, 30, 50 nM) of cadmium telluride QDs. Laser scanning microscopy demonstrated a membrane bound localization of QDs. Vacuoles and cell nuclei of adipocytes were spared by QDs. We conclude that QDs were for the first time proven intracellular in adult adipocytes and demonstrate a strong fluorescence signal. Therefore, they may play an essential role for in vivo tracking of fat grafts

Small poly-L-lysines improve cationic lipid-mediated gene transfer in vascular cells in vitro and in vivo

The potential of two small poly-L-lysines ( sPLLs), low molecular weight sPLL ( LMW-L) containing 7 - 30 lysine residues and L18 with 18 lysine repeats, to enhance the efficiency of liposome-mediated gene transfer ( GT) with cationic lipid DOCSPER {[}1,3- dioleoyloxy- 2-( N-5-carbamoyl-spermine)-propane] in vascular smooth muscle cells ( SMCs) was investigated. Dynamic light scattering was used for determination of particle size. Confocal microscopy was applied for colocalization studies of sPLLs and plasmid DNA inside cells. GT was performed in proliferating and quiescent primary porcine SMCs in vitro and in vivo in porcine femoral arteries. At low ionic strength, sPLLs formed small complexes with DNA ( 50 100 nm). At high ionic strength, large complexes ( 11 mu m) were observed without any significant differences in particle size between lipoplexes ( DOCSPER/ DNA) and lipopolyplexes ( DOCSPER/ sPLL/ DNA). Both sPLLs were colocalized with DNA inside cells 24 h after transfection, protecting DNA against degradation. DOCSPER/ sPLL/ DNA formulations enhanced GT in vitro up to 5- fold, in a porcine model using local periadventitial application up to 1.5- fold. Both sPLLs significantly increased liposome- mediated GT. Poly-L-lysine L18 was superior to LMW-L since it enabled maximal GT at a 10-fold lower concentration. Thus, sPLLs may serve as enhancers for GT applications in SMCs in vitro and in vivo using local delivery. Copyright (c) 2007 S. Karger AG, Basel

Epidermal Growth Factor–PEG Functionalized PAMAM-Pentaethylenehexamine Dendron for Targeted Gene Delivery Produced by Click Chemistry

Aim of this study was the site-specific conjugation of an epidermal growth factor (EGF)-polyethylene glycol (PEG) chain by click chemistry onto a poly(amido amine) (PAMAM) dendron, as a key step toward defined multifunctional carriers for targeted gene delivery. For this purpose, at first propargyl amine cored PAMAM dendrons with ester ends were synthesized. The chain terminal ester groups were then modified by oligoamines with different secondary amino densities. The oligoamine-modified PAMAM dendrons were well biocompatible, as demonstrated in cytotoxicity assays. Among the different oligoamine-modified dendrons, PAMAM-pentaethylenehexamine (PEHA) dendron polyplexes displayed the best gene transfer ability. Conjugation of PAMAM-PEHA dendron with PEG spacer was conducted via click reaction, which was performed before amidation with PEHA. The resultant PEG-PAMAM-PEHA copolymer was then coupled with EGF ligand. pDNA transfections in HuH-7 hepatocellular carcinoma cells showed a 10-fold higher efficiency with the polyplexes containing conjugated EGF as compared to the ligand-free ones, demonstrating the concept of ligand targeting. Overall gene transfer efficiencies, however, were moderate, suggesting that additional measures for overcoming subsequent intracellular bottlenecks in delivery have to be taken

Tissue-Specific Gene Delivery via Nanoparticle Coating

Author Manuscript: 2010 August 1.The use of biomaterials for gene delivery can potentially avoid many of the safety concerns with viral gene delivery. However, the efficacy of polymeric gene delivery methods is low, particularly in vivo. One significant concern is that the interior and exterior composition of polymeric gene delivery nanoparticles are often coupled, with a single polymer backbone governing all functions from biophysical properties of the polymer/DNA particle to DNA condensation and release. In this work we develop electrostatically adsorbed poly(glutamic acid)-based peptide coatings to alter the exterior composition of a core gene delivery particle and thereby affect tissue-specificity of gene delivery function in vivo. We find that with all coating formulations tested, the coatings reduce potential toxicity associated with uncoated cationic gene delivery nanoparticles following systemic injection. Particles coated with a low 2.5:1 peptide:DNA weight ratio (w/w) form large 2 μ sized particles in the presence of serum that can facilitate specific gene delivery to the liver. The same particles coated at a higher 20:1 w/w form small 200 nm particles in the presence of serum that can facilitate specific gene delivery to the spleen and bone marrow. Thus, variations in nanoparticle peptide coating density can alter the tissue-specificity of gene delivery in vivo.National Institutes of Health (U.S.) (BRP: 1R01CA124427-01)National Institutes of Health (U.S.) (EB 000244)National Institutes of Health (U.S.) (U54 CA119349-01)David & Lucile Packard Foundation (Fellowship 1999-1453A

Targeted Disruption of the PME-1 Gene Causes Loss of Demethylated PP2A and Perinatal Lethality in Mice

Phosphoprotein phosphatase 2A (PP2A), a major serine-threonine protein phosphatase in eukaryotes, is an oligomeric protein comprised of structural (A) and catalytic (C) subunits to which a variable regulatory subunit (B) can associate. The C subunit contains a methyl ester post-translational modification on its C-terminal leucine residue, which is removed by a specific methylesterase (PME-1). Methylesterification is thought to control the binding of different B subunits to AC dimers, but little is known about its physiological significance in vivo.Here, we show that targeted disruption of the PME-1 gene causes perinatal lethality in mice, a phenotype that correlates with a virtually complete loss of the demethylated form of PP2A in the nervous system and peripheral tissues. Interestingly, PP2A catalytic activity over a peptide substrate was dramatically reduced in PME-1(-/-) tissues, which also displayed alterations in phosphoproteome content.These findings suggest a role for the demethylated form of PP2A in maintenance of enzyme function and phosphorylation networks in vivo

SIK1/SOS2 networks: decoding sodium signals via calcium-responsive protein kinase pathways

Changes in cellular ion levels can modulate distinct signaling networks aimed at correcting major disruptions in ion balances that might otherwise threaten cell growth and development. Salt-inducible kinase 1 (SIK1) and salt overly sensitive 2 (SOS2) are key protein kinases within such networks in mammalian and plant cells, respectively. In animals, SIK1 expression and activity are regulated in response to the salt content of the diet, and in plants SOS2 activity is controlled by the salinity of the soil. The specific ionic stress (elevated intracellular sodium) is followed by changes in intracellular calcium; the calcium signals are sensed by calcium-binding proteins and lead to activation of SIK1 or SOS2. These kinases target major plasma membrane transporters such as the Na+,K+-ATPase in mammalian cells, and Na+/H+ exchangers in the plasma membrane and membranes of intracellular vacuoles of plant cells. Activation of these networks prevents abnormal increases in intracellular sodium concentration

Targeted gene delivery in tumor xenografts by the combination of ultrasound-targeted microbubble destruction and polyethylenimine to inhibit survivin gene expression and induce apoptosis

<p>Abstract</p> <p>Background</p> <p>Noninvasive and tissue-specific technologies of gene transfection would be valuable in clinical gene therapy. This present study was designed to determine whether it could enhance gene transfection <it>in vivo </it>by the combination of ultrasound-targeted microbubble destruction (UTMD) with polyethylenimine (PEI) in tumor xenografts, and illuminate the effects of gene silencing and apoptosis induction with short hairpin RNA (shRNA) interference therapy targeting human survivin by this novel technique.</p> <p>Methods</p> <p>Two different expression vectors (pCMV-LUC and pSIREN) were incubated with PEI to prepare cationic complexes (PEI/DNA) and confirmed by the gel retardation assay. Human cervical carcinoma (Hela) tumors were planted subcutaneously in both flanks of nude mice. Tumor-bearing mice were administered by tail vein with PBS, plasmid, plasmid and SonoVue microbubble, PEI/DNA and SonoVue microbubble. One tumor was exposed to ultrasound irradiation, while the other served as control. The feasibility of targeted delivery and tissue specificity facilitated by UTMD and PEI were investigated. Moreover, immunohistochemistry analyses about gene silencing and apoptosis induction were detected.</p> <p>Results</p> <p>Electrophoresis experiment revealed that PEI could condense DNA efficiently. The application of UTMD significantly increases the tissue transfection. Both expression vectors showed that gene expressions were present in all sections of tumors that received ultrasound exposure but not in control tumors. More importantly, the increases in transgene expression were related to UTMD with the presence of PEI significantly. Silencing of the survivin gene could induce apoptosis effectively by downregulating survivin and bcl-2 expression, also cause up-regulation of bax and caspase-3 expression.</p> <p>Conclusions</p> <p>This noninvasive, novel combination of UTMD with PEI could enhance targeted gene delivery and gene expression in tumor xenografts at intravenous administration effectively without causing any apparently adverse effect, and might be a promising candidate for gene therapy. Silencing of survivin gene expression with shRNA could be facilitated by this non-viral technique, and lead to significant cell apoptosis.</p