Promoter effects of adeno-associated viral vector for transgene expression in the cochlea in vivo

The aims of this study were to evaluate the expression of enhanced green fluorescent protein (EGFP) driven by 6 different promoters, including cytomegalovirus IE enhancer and chicken beta-actin promoter (CAG), cytomegalovirus promoter (CMV), neuron-specific enolase promoter (NSE), myosin 7A promoter (Myo), elongation factor 1alpha promoter (EF-1alpha), and Rous sarcoma virus promoter (RSV), and assess the dose response of CAG promoter to transgene expression in the cochlea. Serotype 1 adeno-associated virus (AAV1) vectors with various constructs were transduced into the cochleae, and the level of EGFP expression was examined. We found the highest EGFP expression in the inner hair cells and other cochlear cells when CAG promoter was used. The CMV and NSE promoter drove the higher EGFP expression, but only a marginal activity was observed in EF-1alpha promoter driven constructs. RSV promoter failed to driven the EGFP expression. Myo promoter driven EGFP was exclusively expressed in the inner hair cells of the cochlea. When driven by CAG promoter, reporter gene expression was detected in inner hair cells at a dose as low as 3x10(7) genome copies, and continued to increase in a dose-dependent manner. Our data showed that individual promoter has different ability to drive reporter gene expression in the cochlear cells. Our results might provide important information with regard to the role of promoters in regulating transgene expression and for the proper design of vectors for gene expression and gene therapy.     

Efficiency of DNA Transfection of Rat Heart Myoblast Cells H9c2(2-1) by Either Polyethyleneimine or Electroporation

Expression of exogenous DNA in vitro is significantly affected by the particular transfection method utilized. In this study, we evaluated the efficiency of two transfection methods, chemically mediated polyethyleneimine (PEI) treatment and physically mediated electroporation, on a rat heart myoblast cell line, H9c2(2-1). After PEI transfection of pPgk-1/EGFP into H9c2(2-1) cells, EGFP expression could be easily detected by fluorospectrometer after 48 h (210 ± 12 RFU) and continued to increase after 72 h (243 ± 14 RFU). However, when H9c2(2-1) cells were transfected by electroporation (200 V, 500 μF, and one pulse), low level EGFP expression was observed after 48 h (49 ± 4 RFU) or 72 h (21 ± 14 RFU). In contrast, the easily transfectable control CHO-K1 cell line displayed a stronger EGFP expression than the H9c2(2-1) cells either by PEI or electroporation transfection. When transfection efficiencies were assayed by flow cytometry after 72 h, 13.6 ± 2.2% of PEI and 10.1 ± 1.5% of electroporation (250 V, 500 μF, and two pulses) transfected cells of H9c2(2-1) expressed EGFP, and PEI-transfected cells appeared to be less damaged (viability 93.6%) as compared to electroporation-transfected cells (39.5%). However, both PEI and electroporation (580 V, 50 Ω, and 50 μF) were effective for transfection of CHO-K1 with a higher efficiency, cell viability, and EGFP expression than H9c2(2-1). Our results indicate that the transfection efficiency of different methods varies among cell lines and that PEI is more efficient than electropolation for transfection of H9c2(2-1) whereas both PEI and electroporation are suitable for CHO-K1 transfection.     

Layer-by-layer assembly of small interfering RNA and poly(ethyleneimine) for substrate-mediated electroporation with high efficiency

Electroporation microarrays have been developed for the high-throughput transfection of expression constructs and small interfering RNAs (siRNAs) into living mammalian cells. These techniques have potential to provide a platform for the cell-based analysis of gene functions. One of the key issues associated with microarray technology is the efficiency of transfection. The capability of attaining reasonably high transfection efficiency is the basis for obtaining functional data without false negatives. In this study, we aimed at improving the transfection efficiency in the system that siRNA loaded on an electrode is electroporated into cells cultured directly on the electrode. The strategy we adopted here is to increase the surface density of siRNA loaded onto electrodes. For this purpose, the layer-by-layer assembly of siRNA and cationic polymers, branched or linear form of poly(ethyleneimine), was performed. The multilayer thus obtained was characterized by infrared reflection-adsorption spectroscopy and surface plasmon resonance analysis. Transfection efficiency was evaluated in a system that siRNA specific for enhanced green fluorescent protein (EGFP) was electroporated on the electrode into human embryonic kidney cells stably transformed with the EGFP gene. The suppression of EGFP expression was assessed by fluorescence microscopy and flow cytometry. Our data showed that the layer-by-layer assembly of siRNA with branched poly(ethyleneimine) facilitated to increase the surface density of loaded siRNA. As a result, the expression of EGFP gene in the electroporated cells was suppressed much more on the electrodes with the multilayer of siRNA than that with the monolayer.     

Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening

Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC–OCT4–EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC–AAVS1–EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC–AAVS1–EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes.     

Establishment of a Tumor-bearing Mouse Model Stably Expressing Human Tumor Antigens Survivin and MUC1 VNTRs

The eukaryotic vectors VR1012 expressing survivin or 33 tandem repeats of human mucin 1(MUC1)(VNTRs),namely,VR1012-S and VR1012-VNTR(VNTR=variable number of tandem repeat),were constructed by cloning s...     

Photochemical internalization enhances the cytotoxic effect of the protein toxin gelonin and transgene expression in sarcoma cells

Further advantages in the treatment of soft-tissue sarcomas will only be achieved by tailoring the adjuvant therapy after surgery. The photochemically directed release of macro-molecules from endosomes and lysosomes into the cytosol is a novel technology, named photochemical internalization (PCI), that has been evaluated for treatment of sarcoma cells in vitro. Two human synovial sarcoma cell lines (SW 982 and CME-1) were treated with the photosensitizer meso-tetraphenylporphine with two sulfonate groups on adjacent phenyl rings (TPPS2a) and a plasmid encoding enhanced green fluorescent protein (EGFP) complexed to poly-L-lysine to investigate the influence of PCI on gene transfer and with 5 micrograms/mL gelonin to investigate PCI of a Type-I ribosome-inactivating protein toxin. In addition, both cell lines were transduced with an Adenovirus serotype 5 encoding the Escherichia coli lacZ gene (AdHCMV-lacZ, expressing beta-galactosidase) and treated with TPPS2a and light to evaluate the effect of PCI on the transduction rate. Photochemically induced transfection with the reporter gene EGFP in CME-1 cells increased from 0% of cells at no light to 40% of the cells after 60 s of light exposure. In contrast, the SW 982 cells showed no enhanced expression of the gene. The fraction of virally transduced cells was about doubled in both cell lines by means of PCI, although the transduction was more efficient in the CME-1 cells. Both cell lines became up to four-fold more sensitive to light when combining photochemical treatment with gelonin incubation. Our experiments showed that PCI induced the endocytic escape of therapeutic substances in cells derived from human soft-tissue sarcomas.     

Analysis of affinity maps of membrane proteins on individual human embryonic stem cells

The heterogeneity found in many cell types has greatly inspired research in single-cell gene and protein profiling for discovering the origin of heterogeneity and its role in cell fate decisions. Among the existing techniques to probe heterogeneity, atomic force microscopy (AFM) utilizes an antibody/ligand-modified tip to explore the distribution of a target membrane protein on individual cells in their native environment. In this paper, we establish a practical model to analyze the data systematically, and attempt the quantification of membrane protein abundance on single cells by taking account issues, such as the level of nonspecific interaction, the probe resolution, and the reproducibility of detecting protein distribution. We demonstrated the application in examining the heterogeneous distribution and the local protein abundance of TRA-1-81 antigen on human embryonic stem (hES) cells at the subcellular level. Heterogeneity in TRA-1-81 expression was also detected at the single cell level, suggesting the presence of subpopulation cells within an undifferentiated hES cell colony. The method provides a platform to unveiling the correlation between heterogeneity of membrane proteins and cell development in a complex cell community.     

Monitoring transfected cells without selection agents by using the dual-cassette expression EGFP vectors

Conventional methods of selecting gene transfected cells by toxic agents may yield ambiguous results. It is difficult to determine whether cell death is due to selection agents or gene transfection, owing to the substantial overlap of the time-courses for both effects. Therefore, to determine transfection-induced cell toxicity, the mammalian expression vector pEGFP-N1 (CLONTECH Lab., Palo Alto, CA, USA) has been modified to the dual-cassette expression vectors named pEGFP-Ks by the relocation of its EGFP expression cassette. We have precisely monitored the cells transfected with this vector on our custom culture dishes, thereby bypassing the need for selection agent or fluorescent cell sorting. This is a useful method to screen genes encoding potential toxic or useful proteins without performing undesirable selection agent and also can be used to monitor the transfected cells for various purposes, either the inhibition or proliferation of mammalian cells for applications in biotechnology.     

Establishment of a Tumor-bearing Mouse Model Stably Expressing EGFP Labeled Human MUC1 VNTRs

Two eukaryotic vectors expressing 9 tandem repeats of human MUCI（VNTR）, VR1012-VNTR, and pEGFP-VNTR, were constructed by cloning VNTR gene into VR1012 and pEGFP, respectively. VNTR stably expressing murine Lewis lung carcinoma（LLC） cell line（VNTR^＋ LLC） was established by Lipofectamine-mediated transfection of pEGFP-VNTR into LLC cells. The EGFP expression was observed under a fluorescent microscope and VNTR expression in VNTR^＋ LLC cells was confirmed by means of Western blotting. A syngenic graft tumor model was generated by subcutaneous injection of VNTR^＋ LLC cells into C57/BL6 mice and tumor size increased rapidly with time and in a cell qumber dependent manner. VNTR mRNA expression in the tumor formed was confirmed by RT-PCR. After the third immunization mice were challenged subcutaneously with 5×10^5 VNTR^＋ LLC cells, a significant reduction of subcutaneous tumor growth was observed in the groups immunized with VNTR plasmid DNA compared with that in the groups immunized with the vector DNA alone. Thus, the suppression of subcutaneous tumor was antigen-specific. This model is useful for the development of tumor vaccines targeting MUCI VNTRs.     

Dynamic changes of gangliosides expression during the differentiation of embryonic and mesenchymal stem cells into neural cells

Stem cells are used for the investigation of developmental processes at both cellular and organism levels and offer tremendous potentials for clinical applications as an unlimited source for transplantation. Gangliosides, sialic acid-conjugated glycosphingolipids, play important regulatory roles in cell proliferation and differentiation. However, their expression patterns in stem cells and during neuronal differentiation are not known. Here, we investigated expression of gangliosides during the growth of mouse embryonic stem cells (mESCs), mesenchymal stem cells (MSCs) and differentiated neuronal cells by using high-performance thin-layer chromatography (HPTLC). Monosialoganglioside 1 (GM1) was expressed in mESCs and MSCs, while GM3 and GD3 were expressed in embryonic bodies. In the 9-day old differentiated neuronal cells from mESCs cells and MSCs, GM1 and GT1b were expressed. Results from immunostaining were consistent with those observed by HPTLC assay. These suggest that gangliosides are specifically expressed according to differentiation of mESCs and MSCs into neuronal cells and expressional difference of gangliosides may be a useful marker to identify differentiation of mESCs and MSCs into neuronal cells.     

Bmp 2 and Bmp 7 Induce Odonto- And Osteogenesis of Human Tooth Germ Stem Cells

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain odontogenesis and osteogenesis. In this study, we studied the effect of bone morphogenic protein 2 (BMP 2) and bone morphogenic protein 7 (BMP 7) as differentiation inducers in tooth and bone regeneration. We compared the effect of BMP 2 and BMP 7 on odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs). Third molar-derived hTGSCs were characterized with mesenchymal stem cell surface markers by flow cytometry. BMP 2 and BMP 7 were transfected into hTGSCs and the cells were seeded onto six-well plates. One day after the transfection, hTGSCs were treated with odontogenic and osteogenic mediums for 14 days. For confirmation of odontogenic and osteogenic differentiation, mRNA levels of BMP2, BMP 7, collagen type 1 (COL1A), osteocalsin (OCN), and dentin sialophosphoprotein (DSPP) genes were measured by quantitative real-time PCR. In addition to this, immunocytochemistry was performed by odontogenic and osteogenic antibodies and mineralization obtained by von Kossa staining. Our results showed that the BMP 2 and BMP 7 both promoted odontogenic and osteogenic differentiation of hTGSCs. Data indicated that BMP 2 treatment and BMP 7 treatment induce odontogenic differentiation without affecting each other, whereas they induce osteogenic differentiation by triggering expression of each other. These findings provide a feasible tool for tooth and bone tissue engineering.     

Genetic and expression alterations in association with the sarcomatous change of cholangiocarcinoma cells

Cholangiocarcinoma (CC) is an intrahepatic bile duct carcinoma with a high mortality rate and a poor prognosis. Sarcomatous change/epithelial mesenchymal transition (EMT) of CC frequently leads to aggressive intrahepatic spread and metastasis. The aim of this study was to identify the genetic alterations and gene expression pattern that might be associated with the sarcomatous change in CC. Previously, we established 4 human CC cell lines (SCK, JCK1, Cho-CK, and Choi-CK). In the present study, we characterized a typical sarcomatoid phenotype of SCK, and classified the other cell lines according to tumor cell differentiation (a poorly differentiated JCK, a moderately differentiated Cho-CK, and a well differentiated Choi-CK cells), both morphologically and immunocytologically. We further analyzed the genetic alterations of two tumor suppressor genes (p53 and FHIT) and the expression of Fas/FasL gene, well known CC-related receptor and its ligand, in these four CC cell lines. The deletion mutation of p53 was found in the sarcomatoid SCK cells. These cells expressed much less Fas/FasL mRNAs than did the other ordinary CC cells. We further characterize the gene expression pattern that is involved in the sarcomatous progression of CC, using cDNA microarrays that contained 18,688 genes. Comparison of the expression patterns between the sarcomatoid SCK cells and the differentiated Choi-CK cells enabled us to identify 260 genes and 247 genes that were significantly over-expressed and under-expressed, respectively. Northern blotting of the 14 randomly selected genes verified the microarray data, including the differential expressions of the LGALS1, TGFBI, CES1, LDHB, UCHL1, ASPH, VDAC1, VIL2, CCND2, S100P, CALB1, MAL2, GPX1, and ANXA8 mRNAs. Immunohistochemistry also revealed in part the differential expressions of these gene proteins. These results suggest that those genetic and gene expression alterations may be relevant to the sarcomatous change/EMT in CC cells.     

Effect of mammalian cell differentiation on response to exogenous 5-aminolevulinic acid

Many different types of mammalian cells accumulate fluorescing and photosensitizing concentrations of protoporphyrin IX (PpIX) when exposed to exogenous 5-aminolevulinic acid (ALA) in vivo or in vitro. Most types of malignant cells accumulate substantially more ALA-induced PpIX than do the normal cells from which they arose. Most types of malignant cells also are less differentiated than their normal counterparts. We therefore considered the possibility that malignant cells demonstrate a malignant ALA phenotype (accumulate abnormally large amounts of PpIX when exposed to exogenous ALA) as a direct consequence of their less differentiated state. Human promyelocyte cell line HL-60 and mouse preadipocyte cell line 3T3 L1 were induced to differentiate by exposing them to inducing agents in vitro. The HL-60 cells accumulated less ALA-induced PpIX when differentiated, but the 3T3 L1 cells accumulated more. It appears then that changes in the ALA phenotype with changes in the state of differentiation are cell-type specific. The decreased accumulation of ALA-induced PpIX that accompanied differentiation of the promyelocytic leukemia cells may have clinical application for rapid quantitation of the response of myelocytic leukemia patients to differentiation therapy.     

A microfluidic processor for gene expression profiling of single human embryonic stem cells

The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These 'cell population averaging' data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies.     

PEI-g-MPEG/白细胞介素-10基因传递系统转染背根神经节细胞的体外性能研究

探索非病毒基因载体聚乙二醇-聚乙烯亚胺共聚物(PEI-g-MPEG)介导白细胞介素-10(Interleukin-10,IL-10)体外转染原代培养背根神经节细胞(dorsal root ganglion cells,DRGs)的效果.采用本实验室设计合成的PEI-g-MPEG,与同时携带增强型绿色荧光蛋白报告基因及IL-10基因的真核表达质粒DNA(pDC316-EGFP/IL-10)形成复合物,以脂质体(lipofectamine)复合体系Lipo/pDNA为对照,通过溴乙啶(ethidiumbromide,EB)排斥实验、凝胶阻滞电泳实验、粒径与电位的测定及扫描电镜等实验方法观察PEI-g-MPEG/pDNA的复合效果.并且检测了复合物对DRGs的毒性、转染效果及IL-10的蛋白表达情况.结果表明,PEI-g-MPEG在N/P(PEI-g-MPEG所含的氮原子和质粒DNA中磷原子的摩尔比)为5时可完全复合pDNA;随着N/P的增大,PEI-g-MPEG/pDNA复合物的粒径逐渐减小,而表面电位逐渐增大;在N/P为15时报告基因转染效果和IL-10蛋白表达情况较好,复合物的形貌呈大小均一的球形.PEI-g-MPEG/IL-10基因传递系统对于神经病理性疼痛的基因治疗具有潜在应用价值.     

Silica-based gene reverse transfection: an upright nanosheet network for promoted DNA delivery to cells

A method for substrate-mediated reverse gene transfection was developed using a silica film composed of an upright-sheet network. The silica film with a dense upright-sheet network shows approximately double higher transgene expression efficiency than that of solution-based transfection.     

Laterally stabilized complexes of DNA with linear reducible polycations: strategy for triggered intracellular activation of DNA delivery vectors.

Target-specific DNA delivery requires vectors that combine stability in the biological milieu, receptor-mediated uptake into target cells, and intracellular activation to mediate transgene expression. This is achieved here using polymer-coated vectors based on plasmid DNA complexed with a reductively degradable polycation (RPC), designed for intercellular degradation. The RPC were prepared by oxidation of the terminal cysteinyl thiol groups of Cys(Lys)10Cys. The complexes were coated and surface-cross-linked using multivalent reactive copolymers of N-(2-hydroxypropyl)methacrylamide (PHPMA), providing a unique combination of steric and reversible lateral stabilization, known to promote extended circulation in the bloodstream. Coated complexes containing RPC exhibited lateral stabilization that was reversible by treatment with 2.5 mM dithiothreitol, releasing free DNA after incubation with a polyanion. In contrast, coated complexes containing nonreducible poly(l-lysine) (PLL) were not destabilized by reduction. The biological usefulness of this trigger mechanism was examined by measuring transfection activity in human retinoblast 911 cells of coated complexes, based on PLL or RPC, targeted to cell surface receptors by covalent linkage of basic fibroblast growth factor. The levels of transgene expression observed for RPC-based targeted vectors indicated efficient intracellular activation, authenticating the concept that lateral stabilization introduced by surface coating with PHPMA can be reversed by intracellular reduction.