Development of a laboratory scalable process for enhancing lentivirus production by transient transfection of HEK293 adherent cultures.

Transfection of surface adherent cells remain as a standard methodology for lentiviral production for early phase clinical studies and research purposes. Production today is based on transient co-transfection of three or four plasmids, where the viral elements are encoded separately for safety reasons. Assembly of functional lentiviral particles requires all plasmids to be efficiently transfected into each cell, a notable challenge with many currently available methods for transient transfection. We have previously demonstrated the significant improvement of cationic polymer-based transfection in various cell types using a combination of fusogenic lipids and histone deacetylase 6 inhibitor (Enhancers). In this report, we focused on the transfection step and the feasibility of improving lentiviral production using the Enhancers. After optimization of DNA amount and N/P ratio, transfection using seven commercial gene carriers showed comparable maximal efficiency of production with high cell viability. In the presence of Enhancers, the production of functional lentivirus using LPEI was increased by as much as tenfold and outperformed lentiviral production using Lipofectamine 3000. We demonstrate a scalable and optimized workflow where the use of the Enhancers significantly improved the lentiviral particle production in various HEK293 cell lines.

Thermoporometry characterization of silica microparticles and nanowires.

We present the results of a systematic study on the porosity of silica microparticles and nanowires prepared by glancing angle deposition-metal-assisted chemical etching (GLAD-MACE) and interference lithography-metal-assisted chemical etching (IL-MACE) techniques using the thermoporometry (TPM) method. Good agreement was obtained between our TPM results and published data provided by the suppliers of silica microparticles. TPM characterization of the GLAD-MACE and IL-MACE nanowires was carried out on the basis of parameters obtained from TPM experiments on microparticles. Our nanowires showed a similar trend but lower values of the pore volume and surface area than nanowires prepared by MACE with AgNO3 solution. We attribute the enhanced bioanalysis performance of the GLAD-MACE nanowires based devices to the increased pore volume and total surface area of the nanowires.

The effects of particle size and surface coating on the cytotoxicity of nickel ferrite.

The safety and toxicity of nanoparticles are of growing concern despite their significant scientific interests and promising potentials in many applications. The properties of nanoparticles depend not only on the size but also the structure, microstructure and surface coating. These in turn are controlled by the synthesis and processing conditions. The dependence of cytotoxicity on particle size and on the presence of oleic acid as surfactant on nickel ferrite particles were investigated in vitro using the Neuro-2A cell line as a model. For nickel ferrite particles without oleic acid prepared by ball milling, cytotoxicity was independent of particle size within the given mass concentrations and surface areas accessible to the cells. For nickel ferrite particles coated with oleic acid prepared by the polyol method, the cytotoxicity significantly increased when one or two layers of oleic acid were deposited. Large particles (150+/-50 nm diameter) showed a higher cytotoxicity than smaller particles (10+/-3 nm diameter).

Pharmacological analysis of [3H]-senktide binding to NK3 tachykinin receptors in guinea-pig ileum longitudinal muscle-myenteric plexus and cerebral cortex membranes.

1. The binding properties and pharmacological specificity of the selective NK3 tachykinin receptor agonist [3H))-senktide [( 3H]-succinyl[Asp6,MePhe8] substance P (6-11] have been examined in homogenates of guinea-pig ileum longitudinal muscle-myenteric plexus (LM/MP) and cerebral cortex. 2. Scatchard analysis of saturation binding studies in guinea-pig ileum LM/MP and cerebral cortex membranes indicated that [3H]-senktide bound to a single site with apparent high affinity, KD = 2.21 +/- 0.65 nM; Bmax = 13.49 +/- 0.04 fmol mg-1 protein in ileum and KD = 8.52 +/- 0.45 nM; Bmax = 76.3 +/- 1.6 fmol mg-1 protein in cortex (values are means +/- ranges; n = 2). 3. The pharmacological profile for tachykinins and analogues in displacing [3H]-senktide from ileum membranes was: [MePhe7] neurokinin B greater than neurokinin B (NKB) congruent to senktide greater than eledoisin greater than substance P (SP) greater than neurokinin A(NKA) greater than physalaemin greater than [Sar9,Met(O2)11]SP greater than [Nle10]NKA(4-10) = [Glp6,L-Pro9]-SP(6-11) greater than substance P methyl ester, consistent with [3H]-senktide binding to an NK3 subtype of tachykinin receptor. A similar rank order of affinity was obtained for these peptides in displacing [3H]-senktide from cortex membranes. 4. Several tachykinin receptor agonists were tested for their ability to displace [3H]-senktide from ileal and cortical NK3 binding sites and were found to be either weak displacers (pIC50 less than 5.00) or inactive. 5. The binding of [3H]-senktide to cortex membranes was inhibited by GTP (p1C,0 = 6.49)and GTP-gamma- S (p1C,0 = 6.67) with ATP being at least three orders of magnitude less potent (pIC50 = 3.55). 6. These results indicate that both central and peripheral NK3 receptors share a similar pharmacological specificity and that they may be labelled selectively with the NK3 receptor agonist [3H]-senktide.

Polyethylene glycol modified polyethylenimine for improved CNS gene transfer: effects of PEGylation extent.

Poor solubility of polycation complexes with DNA is one drawback for their in vivo use as gene delivery systems. PEGylation often can improve the solubility of the complexes, minimize their aggregation and reduce their interaction with proteins in the physiological fluid. We investigated in vivo application of polyethylene glycol (PEG) modified polyethylenimine (PEI) for gene expression in the central nervous system. Varied numbers of linear PEG (2 kDa) were grafted to branched PEI (25 kDa) from the average number of PEG per one PEI macromolecule at 1-14.5. While higher degrees of PEG grafting did not improve gene expression, a PEI conjugate with one segment of PEG was able to mediate transgene expression in the spinal cord up to 11-fold higher than PEI homopolymer after intrathecal administration of its DNA complexes into the lumbar spinal cord subarachnoid space. Improved gene expression with this conjugate was observed as well in the brain after the lumbar injection. As assessed in in vitro studies, the PEI conjugate with a low degree of PEG grafting was able to reduce the size of polymer DNA complexes, prevent the aggregation of complexes, decrease the interactions of the complexes with serum proteins, counter the inhibition of serum to gene transfer, and enhance transfection efficiency, although not significant in affecting complex formation and reducing in vitro cell toxicity of PEI. The study provides the in vivo evidence that an appropriate degree of PEG modification is decisive in improving gene transfer mediated by PEGylated polymers.

Evaluation of ultra-thin poly(epsilon-caprolactone) films for tissue-engineered skin.

Various natural and synthetic polymeric materials have been used as scaffold matrices for tissue-engineered skin. However, the commercially available skin replacement products pose problems of poor mechanical properties and immunological rejection. We have thus developed a film of 5 microm thickness, via biaxial stretching of poly(epsilon-caprolactone) (PCL), as a potential matrix for living skin replacements. The aim of this study was to evaluate the feasibility of using biaxially stretched PCL films as matrices for culturing human dermal fibroblasts. For this purpose, we cultured human dermal fibroblasts for 7 days on the films. Glass cover slips and polyurethane (PU) sheets were used as controls. The data from phase contrast light, confocal laser, and scanning electron microscopy suggested that biaxially stretched PCL films support the attachment and proliferation of human dermal fibroblasts. Thymidine-labeling results showed quantitatively that cell proliferation on the PCL films was superior to that on the PU samples. These results indicated that biaxially stretched PCL films supported the growth of human dermal fibroblasts and might have potential to be applied in tissue engineering a dermal equivalent or skin graft.

Identification of mammalian GFRalpha-2 splice isoforms.

Neurturin (NTN) belongs to a structurally related family of bioactive molecules which include glial cell-line derived neutrotrophic factor (GDNF) and perserphin (PSP). NTN exerts its effects through a multicomponent receptor system which include a receptor (GFRalpha-2) and the proto-oncogene c-RET. We report here the identification of three splice isoforms of the GFRalpha-2 receptors (GFRalpha-2a, GFRalpha-2b and GFRalpha-2c) by reverse transcription-PCR (RT-PCR). GFRalpha-2b is a novel splice variant. All three isoforms were found to be expressed in various adult murine tissues as well as in the brain of the newborn human. The identity of these isoforms were further confirmed by the isolation of the gene and the characterisation of the splice junctions.

Cloning of a novel murine isoform of the glial cell line-derived neurotrophic factor receptor.

We report here the cloning of a novel form of the murine glial cell line-derived neurotrophic factor (GDNF) receptor. Northern blot analyses of various mouse tissues, including whole brain, demonstrated the existence of multiple transcripts of GDNF receptor. Screening of an adult mouse liver cDNA library yielded two isoforms of the receptor. One of the forms (alpha) shows a high degree of homology with other mammalian GDNFR-alpha and the other novel form (beta) is identical to the alpha form except for a deletion of five amino acids. These two forms do not share high sequence homologies with the recently isolated neurturin receptor. Both the alpha and beta forms are expressed in various murine tissues but not in muscle.

Immunocytochemical localization of neuromedin K (neurokinin B) in rat spinal ganglia and cord.

Specific antisera directed against substance P and neuromedin K (neurokinin B) have been used in double-label immunofluorescence studies to unambiguously localize these two neuropeptides of the tachykinin family in single tissue sections of rat spinal cord and dorsal root ganglia. Substance P-like immunoreactivity (SPLI) is present but neuromedin K-like immunoreactivity (NMKLI) is undetectable in dorsal root ganglia. Both peptides are present in the spinal cord, but NMKLI is largely restricted to the dorsal gray while SPLI shows a broader distribution. In the spinal gray, NMKLI coexists with SPLI in some, but not all, fibers. While substance P in the dorsal spinal cord is largely of primary afferent origin, neuromedin K appears to originate largely from intrinsic spinal neurons.

Simultaneous extraction of total RNA and peptides from tissues: application to tachykinins.

Methods for extraction and isolation of intact RNA are often laborious, time consuming, and preclude the direct analysis of peptides. Similarly, the conditions for extraction and isolation of peptides are unsuitable for the isolation of intact RNA. Thus, to study changes in the levels of neuropeptides and gene expression of the corresponding mRNAs, separate procedures are required. A simple and rapid method for the simultaneous extraction of RNA and peptides from tissues is described. RNA and peptides are extracted with guanidinium isothiocyanate, followed by delipidation, and peptides are isolated by a simple solid-phase extraction procedure. RNA is isolated by differentially partitioning DNA into an organic phase, followed by precipitation with ethanol. The RNA and peptides isolated by this method are of high yield and quality. Furthermore, this method for RNA isolation is successful and efficient, even with tissues that proved recalcitrant with other procedures, and allows the simultaneous processing of multiple samples. We describe the successful application of this procedure for measuring tachykinins and the corresponding preprotachykinin A mRNA from tissues. Extraction of neuropeptide K, a 36-mer tachykinin, was dramatically more efficient with the present method than other methods in common use.

Heterogeneity of tachykinin-like immunoreactive peptides in rat spinal cord and dorsal root ganglia.

The concentrations of tachykinins in rat spinal cord and dorsal root ganglia (DRGs) were measured using a combination of high performance liquid chromatography (HPLC) and radioimmunoassays (RIAs). Substance P-like immunoreactivity (SPLI) was found to be significantly higher than either substance K-like immunoreactivity (SKLI) or neuromedin K-like immunoreactivity (NMKLI) in both tissues. In the spinal cord, the concentration of SKLI was comparable to that of NMKLI. In DRGs, NMKLI is present at concentrations much lower than those of SKLI or SPLI. In addition to immunoreactive components co-eluting with the three mammalian tachykinins SP, SK and NMK, analyses using reverse-phase HPLC revealed an immunoreactive peak co-eluting with the C-terminal octapeptide of SK (SK3-10), and a yet to be identified peak eluting before SK. This study also demonstrates the use of a novel and highly specific RIA for NMK to measure NMKLI without the need of reverse-phase HPLC.

A novel radioimmunoassay for neuromedin K. I. Absence of neuromedin K-like immunoreactivity in guinea pig ileum and urinary bladder. II. Heterogeneity of tachykinins in guinea pig tissues.

A novel and highly specific radioimmunoassay for the tachykinin peptide neuromedin K (NMK, also known as neurokinin beta, neurokinin B) has been developed and used to determine the distribution of this peptide in extracts of guinea pig tissues. In addition to immunoreactive components coeluting with the 3 mammalian tachykinins, substance P (SP), substance K (SK) and NMK, analyses using reverse-phase HPLC revealed immunoreactive peaks coeluting with the C-terminal octapeptide of SK (SK-(3-10], an N-terminally extended form of SK (gamma-preprotachykinin-(72-92)amide), and a yet unidentified peak eluting before NMK in the extracts of guinea pig brain and spinal cord. In contrast to the other tachykinins, SP and SK, which were present in high concentrations in extracts of all peripheral and central tissues examined, NMK-like immunoreactivity was detected only in extracts of central tissues. NMK-like immunoreactivity was not detected in extracts of terminal ileum and urinary bladder.

Presence and actions of vasopressin-like peptides in the rabbit anterior uvea.

The presence of vasopressin-like immunoreactivity (VP-IR) in the rabbit eye was demonstrated by radioimmunoassay. Trigeminal nerve denervation resulted in a significant and selective decrease in the levels of VP-IR in the iris sphincter muscle and the cornea. The isolated iris sphincter muscle contracted in response to low concentrations of [Arg8]vasopressin (AVP) and related peptides. The V1 vasopressin receptor antagonist, d(CH2)5Tyr(Me)AVP, potently inhibited the contractile responses to AVP. AVP was found to induce an increase in the accumulation of inositol phosphates in the iris sphincter muscle but not in the dilator/ciliary body preparation in vitro. The present investigation demonstrates the presence of VP-IR in the rabbit eye and that this substance may be another sensory nerve-derived mediator acting on specific target sites in the anterior uvea.

Preprotachykinin-A and substance P receptor (NK1) gene expression in rat astrocytes in vitro.

The presence of mRNA transcripts coding for preprotachykinin-A and the substance P receptor in cultured astrocytes is demonstrated by a combination of reverse transcription/polymerase chain reaction (PCR) and Southern blotting. These findings showed that astrocytes in culture are capable of synthesing both the precursor of substance P (preprotachykinin-A) and the cognate receptor, substance P receptor (NK1). The simultaneous presence of both the ligand (substance P) and the receptor (NK1) may indicate an autocrine nature of astrocyte communication.

Quantification of mouse glial cell-line derived neurotrophic factor family receptor alpha 2 alternatively spliced isoforms by real time detection PCR using SYBR Green I.

Neurturin (NTN) belongs to the glial cell-line derived neurotrophic factor (GDNF) family of growth factors. Both NTN and GDNF have been shown to potently prevent the degeneration of dopaminergic neuron in vitro and in vivo. The GDNF family receptor alpha 2 (GFR alpha-2) is the preferred receptor for NTN. In addition to the known full-length isoform (GFR alpha-2a), we have previously reported the isolation of two novel alternatively spliced isoforms (GFR alpha-2b and GFR alpha-2c). All three isoforms are expressed in all mammalian tissues examined, including human fetal brain. However, the expression levels of these isoforms have yet to be quantified. In this report, we have developed a real time polymerase chain reaction (PCR) detection method using SYBR Green I to detect the expression levels of the three splice variants (GFR alpha-2a, GFR alpha-2b and GFR alpha-2c). Of the three isoforms, GFR alpha-2a was found to be the most abundant receptor expressed in the whole murine brain. The real time PCR detection method using SYBR Green I developed in this report can be used to unambiguously quantitate expression levels of the GFR alpha-2 isoforms and can be extended to the quantitation of other alternatively spliced isoforms.

Nanostructured Si-nanowire microarrays for enhanced-performance bio-analytics.

We demonstrate the fabrication of a novel platform based on Si nanowire arrays integrated with a programmable DNA-directed homogeneous-phase analyte-capture strategy for robust detection of bio-analytes. The nanofabrication process used, based on a combination of glancing-angle-deposition and metal-assisted-catalytic-etching, is capable of producing thousands of testing sites per chip, and the sites can be fabricated over entire wafers, with precise control of size and positioning, using conventional microelectronics technology. The analyte-capture strategy used eliminates the well-known interference of the heterogeneous-phase (substrate) with the capturing of analytes. We examine the effects of the nanoscale features of the substrates (nanowire porosity and clumping) on the coupling efficiency of analytes and show that the fabricated microarrays are robust, have high efficiency and capacity, and provide significantly enhanced signal-to-noise ratio detection.

snoU6 and 5S RNAs are not reliable miRNA reference genes in neuronal differentiation.

Accurate profiling of microRNAs (miRNAs) is an essential step for understanding the functional significance of these small RNAs in both physiological and pathological processes. Quantitative real-time PCR (qPCR) has gained acceptance as a robust and reliable transcriptomic method to profile subtle changes in miRNA levels and requires reference genes for accurate normalization of gene expression. 5S and snoU6 RNAs are commonly used as reference genes in microRNA quantification. It is currently unknown if these small RNAs are stably expressed during neuronal differentiation. Panels of miRNAs have been suggested as alternative reference genes to 5S and snoU6 in various physiological contexts. To test the hypothesis that miRNAs may serve as stable references during neuronal differentiation, the expressions of eight miRNAs, 5S and snoU6 RNAs in five differentiating neuronal cell types were analyzed using qPCR. The stabilities of the expressions were evaluated using two complementary statistical approaches (geNorm and Normfinder). Expressions of 5S and snoU6 RNAs were stable under some but not all conditions of neuronal differentiation and thus are not suitable reference genes. In contrast, a combination of three miRNAs (miR-103, miR-106b and miR-26b) allowed accurate expression normalization across different models of neuronal differentiation.

Creation of nanostructures by interference lithography for modulation of cell behavior.

Emerging evidence of the striking differences that can be induced in the behavior of biological cells through topographical modulation of physically and chemically patterned nanostructured surfaces provides a great impetus for developing novel cellular-scale and sub-cellular-scale nanopatterned substrates and for employing them for exciting new applications in life and medical sciences and biotechnology. However, the lack of availability of cost-effective, large-surface-area nanofabricated substrates of appropriate dimensions and features has proved to be a major impediment for research in this area. Here, we demonstrate a simple and cost-effective method based on interference lithography to produce spatially precise and wide-surface-coverage silicon- and polymer-based nanostructures to study how cells react to nanoscale structures or surfaces.

Polyplex formation between four-arm poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate) and plasmid DNA in gene delivery.

Amphiphilic polyelectrolytes comprising cationic and uncharged hydrophilic segments condensed negatively charged DNA to form a core-shell structure stabilized by a layer of hydrophilic corona chains. At physiological pH, four-arm star-shaped poly(ethylene oxide)-b-poly(2-(diethylamino)ethyl methacrylate) (four-arm PEO-b-PDEAEMA) block copolymer possessed positively charged amine groups that interacted with negatively charged plasmid DNA to form polymer/DNA complexes. The mechanism and physicochemical properties of the complex formation were investigated at varying molar ratio of amine groups on polymer chains and phosphate group on plasmid DNA segments (N/P ratio). The capability of the star block copolymer to condense DNA was demonstrated through gel electrophoresis and ethidium bromide exclusion assay. In the absence of salt, the hydrodynamic radius of polyplexes was about 94 nm at low polymer/DNA ratio, and it decreased to about 34 nm at large N/P ratios, forming a compact spherical structure with a weighted average molecular weight of 4.39 +/- 0.22 x 10(6) g/mol. Approximately 15 polymeric chains were required to condense a plasmid DNA. The addition of monovalent salt to the polyplexes significantly altered the size of the complexes, which would have an impact on cell transfection. Because of the electrostatic interaction induced by the diffusion of small ions, the polyplex increased in size to about 53 nm with a less compact structure. In vitro cytotoxicty of polymer and polymer/pDNA complexes were evaluated, and the polyplexes exhibited low toxicity at low N/P ratios. At N/P ratio of 4.5, the four-arm PEO-b-PDEAEMA showed the highest level of transfection in Neuro-2A cells. These observations showed that the star-shaped multi-arm polymers offers interesting properties in self-association and condensation ability for plasmid DNA and can serve as a nonviral DNA delivery system.

Correlating transfection barriers and biophysical properties of cationic polymethacrylates.

Transfection efficiencies of several polymeric gene carriers were compared and correlated quantitatively to the amounts of cellular accumulation of plasmid DNA and to the expression of mRNA by quantitative real-time polymerase chain reaction (real-time PCR). Three polycations polymers with similar chemical structure were used in this study: poly(dimethylamino)ethyl methacrylate (PDMA) homopolymer, PEO-b-PDMA copolymer, and PEO-b-poly(diethylamino)ethyl methacrylate (PEO-b-PDEA) copolymer. Despite their similar chemical structures, the transfection efficiencies were significantly different. PEO-b-PDEA copolymer was significantly less efficient as gene carrier as compared to both PDMA and PEO-b-PDMA. Correlations between cytotoxicity, cellular uptake of plasmid DNA, expression levels of transgene and protein, and the physical properties of the polymers were observed. With the PEO-b-PDEA studies, cytotoxicity was due primarily to the excess of polymers that did not participate in the DNA binding. In addition, the inability of the polymer/DNA polyplexes to interact with cell effectively was identified as a critical barrier for high efficiency of transfection. This study demonstrated that the use of quantitative real-time PCR in combination with physical characterization techniques could provide useful insights into the transfection barrier at different cellular levels.