Copine1 C2 domains have a critical calcium-independent role in the neuronal differentiation of hippocampal progenitor HiB5 cells

Copine1 (CPNE1) has tandem C2 domains and an A domain and is known as a calcium-dependent membrane-binding protein that regulates signal transduction and membrane trafficking. We previously demonstrated that CPNE1 directly induces neuronal differentiation via Akt phosphorylation in the hippocampal progenitor cell line, HiB5. To determine which region of CPNE1 is related to HiB5 cell neurite outgrowth, we constructed several mutants. Our results show that over-expression of each C2 domain of CPNE1 increased neurite outgrowth and expression of the neuronal marker protein neurofilament (NF). Even though protein localization of the calcium binding-deficient mutant of CPNE1 was not affected by ionomycin, this mutant increased neurite outgrowth and NF expression in HiB5 cells. Furthermore, Akt phosphorylation was increased by over-expression of the calcium binding-deficient CPNE1 mutant. These results suggest that neither cellular calcium levels nor the localization of CPNE1 affect its function in neuronal differentiation. Collectively, our findings indicating that the C2 domains of CPNE1 play a calcium-independent role in regulating the neuronal differentiation of HiB5 cells.     

Copine1 enhances neuronal differentiation of the hippocampal progenitor HiB5 cells

Copine1 is a ubiquitously expressed protein found in various tissues including the brain, but little is known about the physiological function of this protein. Here, we showed that copine1 is involved in neuronal differentiation. Over-expression of copine1 clearly increased neurite outgrowth and expression of Tuj1, a neuronal marker protein, in HiB5 cells. In addition, endogenous copine1 was transiently increased at the early time during neuronal differentiation of HiB5 cells. When the expression of endogenous copine1 was knocked-down by its specific shRNA, PDGF-mediated neurite outgrowth was clearly decreased in HiB5 cells. Furthermore, over-expression of copine1 increased phosphorylation of Akt and copine1-specific shRNA decreased phosphorylation of Akt during neuronal differentiation of HiB5 cells. Interestingly, the phosphorylation level of PI3K, generally known as an upstream protein of Akt, was not changed by copine1 expression. These results suggest that copine1 enhances neuronal differentiation of HiB5 cells not through the PI3K-Akt pathway, but by using another Akt activated signal pathway.     

Differential activation of phospholipases by mitogenic EGF and neurogenic PDGF in immortalized hippocampal stem cell lines

In several neuronal systems, nerve growth factor (NGF) and platelet-derived growth factor (PDGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogenic agent. Hippocampal stem cell lines (HiB5) immortalized by the expression of a temperature-sensitive SV40 large T antigen also respond differentially to EGF and PDGF. While EGF treatment at the permissive temperature induces proliferation, the addition of PDGF induces differentiation at the non-permissive temperature. However, the mechanism responsible for these different cellular fates has not been clearly elucidated. In order to clarify possible critical signaling events leading to these distinct cellular outcomes, we examined whether either EGF or PDGF differentially induces the activation of phospholipases, such as phospholipase A(2) (PLA(2)), C (PLC), or D (PLD). Although EGF stimulation did not induce phospholipases, PDGF caused a rapid and transient activation of PLC and PLD, but not PLA(2). When the activation of PLC or PLD was blocked, the neurite outgrowth induced by PDGF was significantly inhibited. Although the activation of PLD occurred faster than PLC, blocking of PLD activity by transient expression of lipase-inactive mutants did not inhibit the induction of PLC activity by PDGF. These results suggest that the differential activation of phospholipases may play an important role in signal transduction by mitogenic EGF and neurotrophic PDGF in HiB5 neuronal hippocampal stem cells. In particular, the activation of phospholipase C and D may contribute to neuronal differentiation by neurogenic PDGF in the HiB5 cells.     

Immediate transfection of patient-derived leukemia: a novel source for generating cell-based vaccines

Background

The production of cell-based cancer vaccines by gene vectors encoding proteins that stimulate the immune system has advanced rapidly in model systems. We sought to develop non-viral transfection methods that could transform patient tumor cells into cancer vaccines, paving the way for rapid production of autologous cell-based vaccines.

Methods

As the extended culture and expansion of most patient tumor cells is not possible, we sought to first evaluate a new technology that combines electroporation and chemical transfection in order to determine if plasmid-based gene vectors could be instantaneously delivered to the nucleus, and to determine if gene expression was possible in a cell-cycle independent manner. We tested cultured cell lines, a primary murine tumor, and primary human leukemia cells from diagnostic work-up for transgene expression, using both RFP and CD137L expression vectors.

Results

Combined electroporation-transfection directly delivered plasmid DNA to the nucleus of transfected cells, as demonstrated by confocal microscopy and real-time PCR analysis of isolated nuclei. Expression of protein from plasmid vectors could be detected as early as two hours post transfection. However, the kinetics of gene expression from plasmid-based vectors in tumor cell lines indicated that optimal gene expression was still dependent on cell division. We then tested to see if pediatric acute lymphocytic leukemia (ALL) would also display the rapid gene expression kinetics of tumor cells lines, determining gene expression 24 hours after transfection. Six of 12 specimens showed greater than 17% transgene expression, and all samples showed at least some transgene expression.

Conclusion

Given that transgene expression could be detected in a majority of primary tumor samples analyzed within hours, direct electroporation-based transfection of primary leukemia holds the potential to generate patient-specific cancer vaccines. Plasmid-based gene therapy represents a simple means to generate cell-based cancer vaccines and does not require the extensive infrastructure of a virus-based vector system.     

High-throughput screening of soluble recombinant proteins

The aims of high-throughput (HTP) protein production systems are to obtain well-expressed and highly soluble proteins, which are preferred candidates for use in structure-function studies. Here, we describe the development of an efficient and inexpensive method for parallel cloning, induction, and cell lysis to produce multiple fusion proteins in Escherichia coli using a 96-well format. Molecular cloning procedures, used in this HTP system, require no restriction digestion of the PCR products. All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%). To screen for well-expressed soluble fusion protein, total cell lysates of bacteria culture ( approximately 1.5 mL) were subjected to high-speed centrifugation in a 96-tube format and analyzed by multiwell denaturing SDS-PAGE. Our results thus far show that 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs. The method is well suited for automation and is applicable for the production of large numbers of proteins for genome-wide analysis.     

RACK1 identified as the PCBP1‐interacting protein with a novel functional role on the regulation of human MOR gene expression

Poly C binding protein 1 (PCBP1) is an expressional regulator of the mu‐opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co‐regulator modifying human MOR gene expression by protein–protein interaction with PCBP1. A human brain cDNA library was screened using the two‐hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1‐RACK1 interaction was confirmed via in vivo validation using the two‐hybrid system, and by co‐immunoprecipitation with anti‐PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co‐immunoprecipitation suggested that RACK1‐PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over‐expression resulted in a dose‐dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock‐down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT‐PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by ³H‐diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.     

Identification of choriogenin cis-regulatory elements and production of estrogen-inducible, liver-specific transgenic Medaka

Choriogenins (chg-H, chg-L) are precursor proteins of egg envelope of medaka and synthesized in the spawning female liver in response to estrogen. We linked a gene construct chg-L1.5 kb/GFP (a 1.5 kb 5'-upstream region of the chg-L gene fused with a green fluorescence protein (GFP) gene) to another construct emgb/RFP (a cis-regulatory region of embryonic globin gene fused with an RFP gene), injected the double fusion gene construct into 1- or 2-cell-stage embryos, and selected embryos expressing the RFP in erythroid cells. From the embryos, we established two lines of chg-L1.5 kb/GFP-emgb/RFP-transgenic medaka. The 3-month-old spawning females and estradiol-17beta (E2)-exposed males displayed the liver-specific GFP expression. The E2-dependent GFP expression was detected in the differentiating liver of the stage 37-38 embryos. In addition, RT-PCR and whole-mount in situ hybridization showed that the E2-dependent chg expression was found in the liver of the stage 34 embryos of wild medaka, suggesting that such E2-dependency is achieved shortly after differentiation of the liver. Analysis using serial deletion mutants fused with GFP showed that the region -426 to -284 of the chg-L gene or the region -364 to -265 of the chg-H gene had the ability to promote the E2-dependent liver-specific GFP expression of its downstream gene. Further analyses suggested that an estrogen response element (ERE) at -309, an ERE half-site at -330 and a binding site for C/EBP at -363 of the chg-L gene played important roles in its downstream chg-L gene expression. In addition, this transgenic medaka may be useful as one of the test animals for detecting environmental estrogenic steroids.     

Epithelial cell-targeted transgene expression enables isolation of cyan fluorescent protein (CFP)-expressing prostate stem/progenitor cells

To establish a method for efficient and relatively easy isolation of a cell population containing epithelial prostate stem cells, we developed two transgenic mouse models, K5/CFP and K18/RFP. In these models, promoters of the cytokeratin 5 (Krt5) and the cytokeratin 18 (Krt18) genes regulate cyan and red fluorescent proteins (CFP and RFP), respectively. CFP and RFP reporter protein fluorescence allows for visualization of K5⁺ and K18⁺ epithelial cells within the cellular spatial context of the prostate gland and for their direct isolation by FACS. Using these models, it is possible to test directly the stem cell properties of prostate epithelial cell populations that are positively selected based on expression of cytoplasmic proteins, K5 and K18. After validating appropriate expression of the K5/CFP and K18/RFP transgenes in the developing and adult prostate, we demonstrate that a subset of CFP-expressing prostate cells exhibits stem cell proliferation potential and differentiation capabilities. Then, using prostate cells sorted from double transgenic mice (K5/CFP + K18/RFP), we compare RNA microarrays of sorted K5⁺K18⁺ basal and K5⁻K18⁺ luminal epithelial cells, and identify genes that are differentially expressed. Several genes that are over-expressed in K5⁺ cells have previously been identified as potential stem cell markers. These results suggest that FACS isolation of prostate cells from these mice based on combining reporter gene fluorescence with expression of potential stem cell surface marker proteins will yield populations of cells enriched for stem cells to a degree that has not been attained by using cell surface markers alone.     

p62 modulates Akt activity via association with PKCzeta in neuronal survival and differentiation

p62 is a ubiquitously expressed phosphoprotein that interacts with a number of signaling molecules and a major component of neurofibrillary tangles in the brain of Alzheimer's disease patients. It has been implicated in important cellular functions such as cell proliferation and anti-apoptotic pathways. In this study, we have addressed the potential role of p62 during neuronal differentiation and survival using HiB5, a rat neuronal progenitor cell. We generated a recombinant adenovirus encoding T7-epitope tagged p62 to reliably transfer p62 cDNA into the neuronal cells. The results show that an overexpression of p62 led not only to neuronal differentiation, but also to decreased cell death induced by serum withdrawal in HiB5 cells. In this process p62-dependent Akt phosphorylation occurred via the release of Akt from PKCzeta by association of p62 and PKCzeta, which is known as a negative regulator of Akt activation. These findings indicate that p62 facilitates cell survival through novel signaling cascades that result in Akt activation. Furthermore, we found that p62 expression was induced during neuronal differentiation. Taken together, the data suggest p62 is a regulator of neuronal cell survival and differentiation.     

Comparison of different signal peptides for protein secretion in nonlytic insect cell system

Protein expression and secretion in insect cells have been widely studied in the baculovirus-infected insect cell system. In directly transfected insect cells only intracellular expression and purification of recombinant proteins have been studied in detail. To examine multiple recombinant protein variants, easy and fast expression and a purification screening system are required. The aim of this study was to establish an effective and rapid secretion system for human azurocidin using directly transfected insect cells. We also constructed and tested expression vectors possessing heterologous signal peptides derived from human azurocidin, yellow lupin diphosphonucleotide phosphatase/phosphodiesterase (PPD1), and papaya papain IV to secrete yellow lupin and red kidney bean purple acid phosphatases, PPD1, and papain IV. Our results demonstrate that the secretion vectors used here can direct recombinant proteins to the culture medium very effectively, allowing their simple purification on a small/medium scale. Based on secretion and activity analyses it seems that the azurocidin signal peptide is one of the most potent secretion signals.     

The molecular mechanism of NELL2 movement and secretion in hippocampal progenitor HiB5 cells

Neural epidermal growth factor-like protein-like 2 (NELL2) is a secreted glycoprotein that is predominantly expressed in the nervous system, but little is known about the intracellular movement and secretion mechanism of this protein. By monitoring the localization and movements of enhanced green fluorescent protein (EGFP)-labeled NELL2 in living cultured hippocampal neuroprogenitor HiB5 cells, we determined the subcellular localization of NELL2 and its intracellular movement and secretion mechanism. Cterminal EGFP-fused NELL2 showed a typical expression pattern of secreted proteins, especially with respect to its localization in the endoplasmic reticulum, Golgi apparatus, and punctate structures. Vesicles containing NELL2 exhibited bidirectional movement in HiB5 cells. The majority of the vesicles (70.1%) moved in an anterograde direction with an average velocity of 0.454 μm/s, whereas some vesicles (28.7%) showed retrograde movement with an average velocity of 0.302 μm/s. The movement patterns of NELL2 vesicles were dependent upon the presence of microtubules in HiB5 cells. Anterograde movement of NELL2 did not lead to a detectable accumulation of NELL2 in the peripheral region of the cell, indicating that it was secreted into the culture medium. We also showed that the N-terminal 29 amino acids of NELL2 were important for secretion of this protein. Taken together, these results strongly suggest that the N-terminal region of NELL2 determines both the pattern of its intracellular expression and transport of NELL2 vesicles by high-velocity movement. Therefore, NELL2 may affect the cellular activity of cells in a paracrine or autocrine manner.     

Lentiviral vectors enveloped with rabies virus glycoprotein can be used as a novel retrograde tracer to assess nerve recovery in rat sciatic nerve injury models

Retrograde labeling has become the new “gold standard” technique to evaluate the recovery of injured peripheral nerves. In this study, lentiviral vectors with rabies virus glycoprotein envelop (RABV-G-LV) and RFP genes are injected into gastrocnemius muscle to determine the location of RFP in sciatic nerves. We then examine RFP expression in the L4-S1 spinal cord and sensory dorsal root ganglia and in the rat sciatic nerve, isolated Schwann cells, viral dose to expression relationship and the use of RABV-G-LV as a retrograde tracer for regeneration in the injured rat sciatic nerve. VSV-G-LV was used as control for viral envelope specificity. Results showed that RFP were positive in the myelin sheath and lumbar spinal motorneurons of the RABV-G-LV group. RFP gene could be detected both in myelinated Schwann cells and lumbar spinal motor neurons in the RABV-G-LV group. Schwann cells isolated from the RABV-G-LV injected postnatal Sprague Dawley rats were also RFP-gene positive. All the results obtained in the VSV-G-LV group were negative. Distribution of RFP was unaltered and the level of RFP expression increasing with time progressing. RABV-G-LV could assess the amount of functional regenerating nerve fibers two months post-operation in the four models. This method offers an easy-operated and consistent standardized approach for retrograde labeling regenerating peripheral nerves, which may be a significant supplement for the previous RABV-G-LV-related retrograde labeling study.     

Expression screening,protein purification and NMR analysis of human protein domains for structural genomics

Structural genomics, the determination of protein structures on a genome-wide scale, is still in its infancy for eukaryotes due to the number and size of their genes. Low protein expression and solubility of eukaryotic geneproducts are the major bottlenecks in high-throughput (HTP) recombinant protein production with the E. coli expression systems. To circumvent this problem we decided to focus on separate protein domains. We describe here a fast microtiterplate based, expression and solubility screening procedure, using a combination of in vitro and in vivo expression, and purification with nickel-NTA magnetic beads. All steps are optimized for automatic HTP processing using a liquid handling station. Furthermore, large-scale expression and protein purification conditions are optimized, permitting the purification of 24 protein samples per week. We further show that results obtained from the expression screening can be extrapolated to the production of protein samples for NMR. Starting with 81 cloned human protein domains, in vivo expression was detected in 54 cases, and from 28 of those milligrams of protein were purified. An informative HSQC spectrum was recorded for 18 proteins (22%), half of which were indicative of a folded protein. The success rate and quality of the HSQC spectra suggest that the domain approach holds promise for human proteins.     

A novel approach for gene therapy: engraftment of fibroblasts containing the artificial chromosome expression system at the site of inflammation

BACKGROUND: Rheumatoid arthritis is characterized by inflammation of the synovial tissue. High systemic doses are necessary to achieve therapeutic levels of anti-rheumatic drugs in the joints. Gene transfer might provide a more efficient delivery system for genes encoding therapeutic proteins. METHODS: The artificial chromosome expression system (ACE System) is a new non-integrating, non-viral gene expression system which functions like a natural chromosome. This technology offers advantages over current expression systems because it allows stable and predictable expression of proteins encoded by single or multiple genes over long periods of time. We are developing ex vivo gene therapy using murine artificial chromosomes containing a reporter gene (LacZ and red fluorescent protein (RFP)) for local delivery of genes in rats with adjuvant arthritis (AA). RESULTS: The delivery of the intact ACE System into rat fibroblast-like synoviocytes (FLS) and rat skin fibroblasts (RSF) was detected within 24 to 48 h post-transfection. After growing cells under selection, clones expressing LacZ and RFP were identified. Furthermore, we investigated the feasibility of local delivery of a reporter gene to the joints of rats with AA by ex vivo gene therapy. This resulted in engraftment of the injected cells in the synovial tissue microarchitecture and expression of the reporter gene. CONCLUSIONS: This work demonstrates the potential feasibility of treating arthritis and other inflammatory diseases using fibroblasts containing the ACE System as a non-viral vector for gene therapy.     

Serum-free transient protein production system based on adenoviral vector and PER.C6 technology: high yield and preserved bioactivity

Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an DeltaE1/DeltaE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed DeltaE1/DeltaE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The DeltaE1/DeltaE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of DeltaE1/DeltaE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that DeltaE1/DeltaE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.     

Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5′ located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.     

Quantitative comparison of gene co-expression in a bicistronic vector harboring IRES or coding sequence of porcine teschovirus 2A peptide

In biotechnology, simultaneous expression of more than one target gene is often required. Multicistronic vectors encoding several proteins are being actively developed for this purpose. Most often, the commercially available vectors utilize various types of internal ribosomal entry site of the encephalomyocarditis virus (IRES EMCV). However, many researchers consider bicistronic vectors on the basis of sequences that encode self-cleaving 2A peptides more promising. In the work, we compare the efficiency of gene expression in cells transfected with bicistronic constructs bearing either IRES EMCV or the P2A nucleotide sequence corresponding to the porcine teschovirus-1 2A peptide. Efficiency of gene expression was determined in three mammalian cell lines by measurement of co-expression levels of genes coding for RFP and EGFP proteins linked by IRES or P2A sequence. A higher level of the transgene expression was detected in cells transfected with P2A sequence-based genetic constructs.     

Cytotoxic effect of co-expression of human hepatitis A virus 3C protease and bifunctional suicide protein FCU1 genes in a bicistronic vector

Recent reports on various cancer models demonstrate a great potential of cytosine deaminase/5-fluorocytosine suicide system in cancer therapy. However, this approach has limited success and its application to patients has not reached the desirable clinical significance. Accordingly, the improvement of this suicide system is an actively developing trend in gene therapy. The purpose of this study was to explore the cytotoxic effect observed after co-expression of hepatitis A virus 3C protease (3C) and yeast cytosine deaminase/uracil phosphoribosyltransferase fusion protein (FCU1) in a bicistronic vector. A set of mono- and bicistronic plasmid constructs was generated to provide individual or combined expression of 3C and FCU1. The constructs were introduced into HEK293 and HeLa cells, and target protein synthesis as well as the effect of 5-fluorocytosine on cell death and the time course of the cytotoxic effect was studied. The obtained vectors provide for the synthesis of target proteins in human cells. The expression of the genes in a bicistronic construct provide for the cytotoxic effect comparable to that observed after the expression of genes in monocistronic constructs. At the same time, co-expression of FCU1 and 3C recapitulated their cytotoxic effects. The combined effect of the killer and suicide genes was studied for the first time on human cells in vitro. The integration of different gene therapy systems inducing cell death (FCU1 and 3C genes) in a bicistronic construct allowed us to demonstrate that it does not interfere with the cytotoxic effect of each of them. A combination of cytotoxic genes in multicistronic vectors can be used to develop pluripotent gene therapy agents.