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At least 235 records · Page 13Linked to original sources
PubMed2003

Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons.

Analysis of gene regulatory sequences in primary cultures of neurons has been hampered by inefficient transfection of post-mitotic neurons with reporter plasmids. We describe detailed conditions that allowed a significant improvement of transfection efficiency in primary cultures of serum-supplemented rat fetal hypothalamic cells. Transfected cells expressed the green fluorescent protein (GFP) under the control of the strong but non-cell-specific cytomegalo virus (CMV) promoter or under the thyrotropin-releasing hormone (TRH) promoter, to direct expression only in TRH neurons. Using the CMV promoter-GFP plasmid, we tested several commercially available transfection reagents; the best results were obtained with polyethylenimine (PEI) and Lipofectamine 2000. We optimized the transfection procedure with PEI because it rendered more reproducible results. Transfection with PEI was optimal when cells were transfected at a cellular density of 2.9 x 10(6) cells in 35-mm dishes, with 10 microg of DNA, a PEI/DNA ratio of 8.8 and PEI pH of 6.9. Using these conditions, we were able to detect GFP positive neurons after transfecting the TRH promoter-GFP plasmid. GFP positive cells were successfully purified by FACS. This opens the possibility to use transfection of mammalian CNS post-mitotic neurons for new applications including the purification of specific neuronal subtypes.

3T3 Cells↗
PubMed2001

Intracytoplasmic gene delivery for in vitro transfection with cytoskeleton-specific immunoliposomes.

A novel and highly efficient method of in vitro gene transfection has been developed. This method employs direct intracytoplasmic gene delivery into embryonic cardiocytes using neutral cytoskeletal-antigen specific immunoliposomes (CSIL). These immunoliposomes target cardiocytes specifically under reversible hypoxic conditions. Two independent reporter genes, pGL2 and pSV-beta-galactosidase, were used to verify CSIL-transfection (CSIL-fection). The efficiency of CSIL-fection with firefly luciferase pGL2 vector was 30+ times greater than controls consisting of hypoxic cardiocytes treated with plain liposomes (PL) or normoxic cardiocytes treated with CSIL, PL or naked DNA. CSIL-fection was also compared to cationic liposome transfection. Net cationic liposome transfection appeared to be more efficient than CSIL-fection for pGL2 vectors. However, a smaller number of viable cells was observed in the cationic liposome treated cultures than in the CSIL treated cultures. Therefore, to determine whether more cells were transfected with cationic liposomes or CSIL, pSV-beta-galactosidase vector was used. CSIL-fection with pSV-beta-galactosidase vector produced at least 40 times more transfected cells than those transfected with cationic liposomes. No transfection with pSV-beta-galactosidase vectors was obtained with IgG-liposome, PL or naked DNA treatments. Targeted enhanced efficiency of transfection by this novel method could have practical therapeutic applications in the genetic modification of cells ex vivo that could then be reimplanted into patients for gene therapy.

Animals↗
PubMed2003

Transient transfection protects PC6-3 cells from apoptosis induced by nerve growth factor deprivation.

Some mammalian neurons undergo apoptosis after neurotrophin deprivation. We studied neuronally differentiated PC6-3 pheochromocytoma cells, which are highly dependent on nerve growth factor for survival. We found that transient transfection with green fluorescent protein or beta-galactosidase protected cells from apoptosis induced by nerve growth factor deprivation. Individual transfection reagent components did not produce increased viability of nerve growth factor-deprived cells. This apparent neuroprotective effect from transient transfection was specific to neurotrophin deprivation, as cells treated with H(2)O(2) or staurosporine were not protected. To determine the mechanism of neuroprotection after transfection, the transfection status of identified groups of cells was assessed both before and after nerve growth factor deprivation. The results were consistent with a model whereby cells that are transfected but not yet expressing the transfected protein are relatively protected from nerve growth factor deprivation. We suggest that apoptosis induced by neurotrophin deprivation may interact with processes of transient transfection and expression of foreign genes in neuronal cells. Not only should these interactions be considered in transient transfection studies of neurotrophin-deprived neurons, but also their elucidation could lead to novel methods for achieving neuroprotection.

Animals↗
PubMedDate not supplied

O-Alkyl dioleoylphosphatidylcholinium compounds: the effect of varying alkyl chain length on their physical properties and in vitro DNA transfection activity.

1,2-Dioleoyl-sn-3-ethylphosphocholine (EDOPC) has been previously shown be a highly effective DNA transfection reagent in vitro. To assess the effect of alkyl chain length on transfection efficiency, the O-methyl, O-propyl, O-hexyl, O-decyl, and O-octadecyl derivatives have been prepared from dioleoylphosphatidylcholine using the corresponding alkyl trifluoromethylsulfonate. The methyl, ethyl, and propyl derivatives formed liposomes which were very large and unilamellar. The ethyl and propyl derivatives were equally efficient at mediating transfection (even in the presence of serum) of BHK cells, but the chemically labile methyl derivative was a much weaker transfection agent. The O-decyl and O-octadecyl compounds, which assume the inverted hexagonal phase in excess water (as determined by X-ray diffraction), were almost inactive after manual agitation in both water and in saline; however, after sonication, these compounds exhibited good transfection activity. The O-hexyl derivative displayed novel behavior, assuming the lamellar phase at low and a cubic phase at high ionic strength. All compounds, whether lamellar or not, formed lamellar structures when complexed with DNA. In water, where the hexyl compound dispersed well, sonication diminished transfection activity, whereas at physiological ionic strength, which led to poor manual dispersion, sonication was essential for good transfection. These results emphasize the importance of optimal dispersion of a cationic lipid: too little, and interaction with DNA is handicapped, too much, and the resultant particle transfects poorly. Lipid dispersibility is thus an important variable in assessing lipid transfection agents, and caution is advised in attributing too much significance to chemical structure until interaction with DNA has been optimized.

Alkylation↗
PubMedDate not supplied

Facile and statistical optimization of transfection conditions for secretion of foreign proteins from insect Drosophila S2 cells using green fluorescent protein reporter.

Insect Drosophila melanogaster S2 cells were developed as a plasmid-based and therefore nonlytic expression system for functional foreign proteins. Transfection is an important step to introduce foreign target DNA into cells and should be properly optimized to obtain maximum production yield. Single factor search (SFS) methodology is still generally used to determine optimal condition in a biological system. Although this method is relatively simple to perform, it has many disadvantages such as not considering interactions between several factors and not covering the entire region of the solution pool. Therefore, we approached this optimization problem statistically with response surface (RSM) and evolutionary operation (EVOP) methodologies and compared the transfection efficiencies with the traditional SFS method. We employed secreted green fluorescent protein (GFP) as a reporter for determination of optimal transfection condition and secreted human erythropoietin (hEPO) as a confirming foreign model protein. Consequently, we arrived at the best optimal transient transfection condition (1 microg of plasmid DNA, 5 microg of lipofectin, 2 x 10(6) cells of initial cell number, and 18 h of transfection duration time) through a systematic access in a series of SFS, RSM, and EVOP. The secreted hEPO yield using optimal transient transfection condition by EVOP methodology was enhanced by about 1.8-fold compared to that of traditional SFS. This optimized transient transfection condition can be used as a basis for optimal stable transfections. A linear relationship between secreted GFP fluorescence intensity and secreted hEPO concentration indicated that facile and noninvasive determination of optimal transfection conditions for expression and secretion of foreign proteins in S2 cell cultures was made possible by simple measurement of GFP fluorescence.

Animals↗
PubMedDate not supplied

Assessment of a modular transfection system based upon cellular localization of DNA.

Delivery of plasmid DNA for protein production in mammalian cells is not an efficient process. In this study, the theory that cellular localization of plasmid DNA increases transfection efficiency is examined with an emphasis on the understanding of the cellular association of the components of a ternary transfection complex. Mammalian cells take up transfection reagent-DNA complexes primarily from their local environment. Via formation of a ternary complex consisting of the DNA-transfection reagent complex and a heavy particle, such as silica, the efficiency of transfection is substantially increased. We have analyzed cells transfected with the ternary complexes to determine if sedimentation alone affects the percentage of cells that contain the complexes or specific components of the complex. A significant fraction of cells associate with the ternary complexes, including silica nanoparticles. The percentage of cells that associate with DNA was not significantly influenced by the use of the ternary complex. This result suggests that the silica nanoparticles are more than just a sedimentation agent, being also a secondary transfection reagent. These data also confirm that cells may contain transfection reagent-DNA complex but do not express the protein of interest. This knowledge will be used in further research to better design transfection reagents that will increase the efficiency of protein production.

Animals↗
PubMed2002

Formation of plasmid-based transfection complexes with an acid-labile cationic lipid: characterization of in vitro and in vivo gene transfer.

PURPOSE: This study tests the hypothesis that gene transfer efficiency may be improved through the use of transiently stable transfection complexes that degrade within endosomal compartments and promote plasmid escape into the cytosol. METHOD: An acid labile cationic lipid, O-(2R-1,2-di-O-(1'Z, 9'Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate (BCAT), was designed, synthesized, and tested for enhanced gene transfer activity relative to non-labile controls. RESULTS: The O-alkenyl chains of BCAT were completely hydrolyzed after 4 h incubation in pH 4.5 buffer at 25 degrees C. Addition of BCAT to plasmid DNA in 40% ethanol followed by ethanol evaporation yielded transfection complexes that transfected several cell types in the presence of fetal calf serum and without the need of a helper lipid. Transfection complexes prepared from BCAT displayed higher luciferase expression than the corresponding DCAT complexes (an acid-insensitive derivative of BCAT) for all cell types tested. Uptake studies showed that this increase was not due to a difference in the amount of DNA being delivered. FAGS analysis for GFP expression showed that BCAT transfection complexes yielded 1.6 more transfected cells and 20% higher log mean fluorescence than DCAT transfection complexes. In vivo gene transfer was demonstrated in subcutaneous tumor-bearing mice by systemic administration of a 60 microg plasmid dose. Expression was observed in the lungs and in the tumor, with the highest activity being observed in the lungs. CONCLUSIONS: Our results show that increased transfection can be obtained by coupling the cationic headgroup to the hydrophobic amphiphilic tails via acid-labile bonds. Acid-catalyzed release of the alkyl chains should facilitate dissociation of the cationic lipid headgroup from the plasmid, thus accelerating one of the rate-limiting steps in cationic lipid mediated transfection.

3T3 Cells↗
PubMed2003

In vitro gene transfection in human glioma cells using a novel and less cytotoxic artificial lipoprotein delivery system.

PURPOSE: To develop and evaluate a novel artificial lipoprotein delivery system for in vitro gene transfection in human glioma cells. METHOD: Nanoemulsion was formulated with similar lipid compositions present in natural lipoproteins. The oil phase of nanoemulsion was composed of triolein (70%), egg phosphatidylcholine (22.7%), lysophosphatidylcholine (2.3%), cholesterol oleate (3.0%), and cholesterol (2.0%). To replace the surface protein as in natural lipoprotein, poly-L-lysine was modified to add palmitoyl chains at a basic condition and was incorporated onto the nanoemulsion particles through hydrophobic interaction. A model plasmid DNA, pSV-beta-Gal containing a reporter gene for beta-galactosidase was carried by the nanoemulsion/poly-L-lysine particles. The charge variation of soformed complex was examined by agarose gel electrophoresis and zeta potential measurement. In vitro transfection was conducted on human SF-767 glioma cell line using this new system. After standard X-Gal staining, transfected cells were observed under light microscope. The effect of chloroquine on the transfection was examined and, finally, the cytotoxicity of this new system was evaluated in comparison with commercial Lipofectamine gene transfection system. RESULTS: The plasmid DNA was effectively carried by this artificial lipoprotein delivery system and the reporter gene was expressed in the glioma cells. Transfection efficiency was significantly increased by the treatment of chloroquine, indicating that endocytosis possibly was the major cellular uptake pathway. Compared to Lipofectamine system, this new delivery system demonstrated similar transfection efficiency but a much lower cytotoxicity. In the experiment, the cell viability showed up to 75% using this system compared to only 24% using Lipofectamine system. CONCLUSION: A new artificial lipoprotein delivery system was developed for in vitro gene transfection in tumor cells. The new system showed similar transfection efficiency but a much lower cytotoxicity compared with commercial Lipofectamine system.

Cell Line, Tumor↗
PubMed2004

Biodegradable triblock copolymer of PLGA-PEG-PLGA enhances gene transfection efficiency.

PURPOSE: A tri-block copolymer of PLGA-PEG-PLGA was used as an excipient to enhance the gene transfection efficiency of various cationic polymeric carriers. METHODS: Luciferase plasmid DNA was complexed with polyethylenimine for gene transfection. Various concentrations of PLGA-PEG-PLGA copolymer up to 0.5% were added in the transfection medium to explore whether the copolymer increased the level of gene expression. Pluronic F68 was used as a control. Various polyplexes and different cell lines were used to verify the effect of the triblock copolymer on gene transfection. The cellular uptake extent of radiolabeled plasmid was quantitatively determined as a function of PLGA-PEG-PLGA concentration. RESULTS: PLGA-PEG-PLGA copolymer significantly enhanced gene transfection efficiency at a concentration as low as 0.25% (w/v), which was more effective than Pluronic F68 at the same concentration range. The additive effect of the triblock copolymer in the transfection medium was clearly observed for various cationic polyplexes and cell lines, although the gene expression extents largely depended on polymers and cell lines used. Five- to 10-fold increment of gene transfection levels were attained in the presence of the PLGA-PEG-PLGA tri-block copolymer. The enhanced gene transfection efficiency was attributed to the increased cellular uptake of PEI/DNA complexes in the presence of the PLGA-PEG-PLGA tri-block copolymer. CONCLUSIONS: Biodegradable PLGA-PEG-PLGA tri-block copolymer that facilitates the endocytic process can be used as a novel additive in non-viral gene transfection.

Biocompatible Materials↗
PubMed2000

Transgene expression in human epidermal keratinocytes: cell cycle arrest of productively transfected cells.

We have analysed the consequences of liposome mediated gene transfer into human primary epidermal keratinocytes and compared non-Epstein-Barr Virus (EBV) and EBV based expression vectors that carry the genes encoding human Growth Hormone (hGH) or Enhanced Green Fluorescent Protein (EGFP). Different kinetics between the non-EBV and EBV based vectors were revealed upon subcultivation of hGH transfected keratinocytes. The keratinocytes transfected with non-EBV based vector showed a rapid reduction in hGH production. Although the EBV based vector resulted in more stable expression, this was also reduced over time. Chromatin inactivation by deacetylation was investigated by treatment with sodium butyrate and found not to be the reason for the decreasing expression. Keratinocytes divided into subpopulations enriched for either stem cells or transit amplifying cells, based on beta1-integrin expression and function, do not differ significantly with respect to susceptibility to productive transfection. However, when the keratinocytes were transfected with the EGFP gene and sorted live by FACS into EGFP negative and positive populations, only the negative cells were capable of forming significant numbers of colonies. This is consistent with the observation that the ability to incorporate BrdU was dramatically reduced in the EGFP expressing population within 24-48 h post transfection indicating an almost complete cell cycle arrest. p53 levels were unaffected by the procedures, and the keratinocyte cell line HaCat, mutated in both p53 alleles, also shows a marked reduction in clonogenic potency upon transfection. There was a slight increase of TUNEL positive apoptotic nuclei in the positive population at early time points. However, the apoptotic index was still very low. When we measured the frequency of involucrin expressing cells, we found an increase in the productively transfected population over time indicating an initiation of terminal differentiation. In contrast to the transfected cultures, keratinocytes that were transduced using a retroviral vector showed no decrease in colony forming efficiency. In conclusion we find that transgene expressing cells from transfected cultures of epidermal keratinocytes undergo cell cycle arrest and initiate terminal differentiation by mechanisms which are independent of p53 levels.

Cell Adhesion↗
PubMed2005

Structural aspects of histone H1-DNA complexes and their relation to transfection efficiency.

During transfection, polycation-DNA complexes are normally diluted by the transfection medium, which often contains salt in the physiological concentration range and serum. It is not exactly known to what extent this dilution step influences the properties of the complexes, which in turn influence the transfection efficiency. In order to gain more insight into the size-structure-transfection activity relationship, we prepared histone H1-DNA complexes in NaCl solutions at various concentrations known to determine the size and structure of the resulting complexes. We characterized the complexes by physicochemical methods. Fluorescence correlation spectroscopy enabled relative measurements of complex sizes even under physiological conditions. The different appearances of the complexes were correlated with their transfection efficiency. When transfection was performed by dilution of the complexes in cell-cultivation media, the initial structure of H1-DNA complexes preformed under distinct salt conditions had no significant influence on the transfection efficiency. The dilution of the preformed complexes with cell-cultivation medium resulted in re-formation and aggregation of the complexes. The addition of the complexes to the cells without cell-cultivation medium, however, showed a direct correlation between the size of the complexes and the transfection efficiency (correlation coefficient 0.91). Small complexes did not contribute to the transfection.

Animals↗
PubMed1999

Transfection of small numbers of human endothelial cells by electroporation and synthetic amphiphiles.

OBJECTIVES: This study compared the efficiency of electroporation and synthetic amphiphiles. (SAINT-2pp/DOPE) in transfecting small numbers of human endothelial cells. METHODS AND RESULTS: Optimal transfection conditions were tested and appeared to be 400 V and 960 microF for electroporation and a 10:1 ratio for concentrations of SAINT-2pp/DOPE: plasmid. Using these conditions, cell concentrations were lowered step-wise and we were able to transfect as few as one thousand cells with both methods. For detection of transfection of a small number of cells a sensitive assay was needed (Luciferase). A plasmid containing the neomycin resistance gene was used to determine the transfection rate expressed in colony forming units by counting colonies after selection. At low plasmid concentrations this transfection rate was within the same range for both electroporation and SAINT-2pp/DOPE transfection. Fluorescent in situ hybridisation of metaphase chromosomes of transfected endothelial cells using the plasmid as a probe showed that stable integration was possible with both methods. CONCLUSIONS: Electroporation and a synthetic amphiphile, SAINT-2pp, provide the possibility of transfecting small numbers of cells resulting in stable integration of low plasmid concentrations. The availability of this technology is important in order to obtain functional endothelial cell lines from various human blood vessels for research purposes.

Cell Line, Transformed↗
PubMed1983

Rescue of transforming Epstein-Barr virus (EBV) from EBV-genome-positive epithelial hybrid cells transfected with subgenomic fragments of EBV DNA.

Transfection experiments using subgenomic fragments of the B95-8 strain of Epstein-Barr virus (EBV) DNA and EBV genome (HR-1)-positive epithelial/Burkitt hybrid cells (D98/HR-1) were carried out to determine whether an interaction between the transfecting virus fragment(s) and the endogenous HR-1 EBV genome could take place. Expression of EBV-specific antigens, including early antigen and virus capsid antigen, was examined in transfected cells by immunofluorescence. Attempts were also made to recover biologically active EBV from the D98/HR-1 cells after transfection with cloned fragments of B95-8 DNA. We found that D98/HR-1 cells transfected with the BamHI H or H, F, and X fragments were positive for early antigen 3 days after transfection. Spent media from transfected D98/HR-1 cells maintained for 20-30 days in culture were pooled, filtered, concentrated, and used as a potential source of virus to inoculate human umbilical cord blood lymphocytes. No evidence of transformation was observed with such preparations. However, if spent medium from D98/HR-1 cell cultures was first treated with iododeoxyuridine (to induce EBV DNA synthesis and replicative cycle) and then transfected with the BamHI H, F, and X fragments of B95-8 DNA and used to infect cord blood lymphocytes, transformation was obtained. A lymphoblastoid cell line derived in this manner, designated HI-HFX, is an EBV nuclear antigen-positive nonproducer cell line. Similar results were obtained with preparations from iododeoxyuridine-treated D98/HR-1 cells transfected with the EB 26-36 fragment of B95-8 DNA cloned in a Charon 4A vector. The EB 26-36 fragment contains the BamHI H, F, and X regions.

Cell Transformation, Viral↗
PubMed1988

DNA methylation prevents transfection of genes for specific surface antigens.

Sperm and trophoblast are among the few nucleated human cells that do not express HLA class I antigens. DNA methylation, which is proposed to be a tight mechanism of regulation, may be necessary to turn off these genes. We have investigated the transfectability of HLA class I genes and of the genes for the T-cell differentiation antigens Leu-1 (CD5) and Leu-2 (CD8) in mouse L cells by using human sperm cells and choriocarcinoma cell lines, tumors of trophoblastic origin, as sources of DNA. It was found that DNA from one choriocarcinoma line (JAR) does not transfect genes for HLA, Leu-1, or Leu-2 and that DNA from two other choriocarcinoma lines (BeWo and Ima) transfects only some of the surface markers. Sperm DNA transfects genes for all the surface antigens tested except Leu-1. DNA from control cells and from the line SCH transfects all the markers studied. Southern blots show that all cell types contain apparently intact genes encoding HLA, Leu-1, and Leu-2 and reveal differences in the DNA methylation patterns of genes from different sources of DNA. We treated JAR (the cell line with the lowest transfecting ability) with 5-azacytidine and obtained demethylation of its DNA. This demethylated DNA transfects genes for both HLA class I antigens and Leu-2. Further culture of JAR cells in the absence of 5-azacytidine results in remethylation of their DNA and decreased ability to transfect these surface antigens. These findings indicate that DNA methylation affects the efficiency of transfection of surface antigen genes in L cells.

Cell Line↗
PubMedDate not supplied

Effects of sense tankyrase transfection on smooth muscle cells of corpus cavernosum in rat.

This study was conducted to construct the eukaryotic expression vectors for sense tankyrase and to investigate the effects of tankyrase transfection on smooth muscle cells of corpus cavernosum in the rat. After the eukaryotic expression vectors for sense tankyrase were constructed and identified smooth muscle cells of rat corpus cavernosum were transfected with the recombinant plasmids of sense tankyrase (pcDNA-TNKS). Levels of DNA and RNA were then evaluated. Measurement of telomerase activity was conducted by TRAP-ELISA assay, the length of telomere by Southern blot and the growth curve by MTT assay. We have found that the eukaryotic expression vectors for sense tankyrase were constructed and the recombinant plasmids of sense tankyrase were transfected into smooth muscle cells of rat corpus cavernosum successfully; no significant differences in telomerase activity were observed between TNKS-transfected cells (SMC-TANKS), zero-load- transfected cells (SMC-Zeo), and non-transfected cells (SMC) (P > 0.05). The length of telomere in SMC-TANKS was longer than that in SMC-Zeo or SMC (P < 0.01), and the OD value of TNKS-transfected cell was significantly higher than that of the non-transfected cells (P < 0.01). These results suggested that the eukaryotic expression vectors for sense tankyrase were constructed successfully, which provides the basis for gene therapy. Transfection of recombinant plasmids of sense tankyrase helps change the telomerase length of smooth muscle cells of corpus cavernosum and extend the cell life span.

Animals↗
PubMed1992

Repair of ionizing radiation damage in primate alpha DNA transfected into rat cells.

The time-course of repair of irradiated primate alpha DNA was studied after transfection and recovery from rat NRK cells. Rat cells were chosen for transfection because they have no alpha DNA. Plasmid pBUC4 alpha 10, containing 10 tandem 172 bp alpha DNA subunits in its 5 kbp DNA, was irradiated and introduced into the rat cells by electroporation. The transfected alpha DNA was then recovered from NRK nuclei free of extraneous rat DNA, permitting study of the fate of the transfected alpha DNA in time-course experiments. alpha DNA continuously entered nuclei for processing in the first 2.5 h after transfection. The pool of damaged bases in alpha DNA in NRK nuclei was detectable 2.5 h after transfection. Radiation-induced alpha DNA fragments of electrophoretic mobility intermediate between those of unit nucleotide length were prominent in sequencing gel analyses of alpha DNA for 5-150 min after transfection. These intermediate mobility fragments initially disappeared with T 1/2 of 6-20 min. The alpha DNAs of intermediate mobility are presumed to be intermediates in DNA repair. Residual DNA base damage which had not been processed in the transfected cells could later be unmasked in vitro by conversion to strand breaks by beta-elimination using heat and piperidine or endonuclease III of E. coli. Irradiation of the recipient NRK cells with 5 Gy 4 hours before transfection prolonged the time during which intermediate mobility species could be found, consistent with the increased frequency of intermediate mobility species observed in DNA of monkey CV-1 cells pretreated with small doses of radiation before 300 Gy (Bases et al. 1990).

Animals↗
PubMed2003

Generation of Ag-specific cytotoxic T lymphocytes by DC transfected with in vitro transcribed influenza virus matrix protein (M1) mRNA.

BACKGROUND: Application of DC transfected with tumor Ag RNA is promising for DC-based tumor immunotherapy. In this study, Ag-specific cytotoxic T lymphocytes (CTL) were generated by priming lymphocytes with DC transfected with in vitro transcribed (IVT) influenza virus matrix protein M1 (M1) mRNA. METHODS: Human UC blood-CD34+ cell-derived DC were transfected with IVT mRNA encoding either the enhanced green fluorescence protein (EGFP), or M1 by square-wave electroporation. DC were confirmed to have typical morphology and phenotype. DC transfected with IVT EGFP mRNA were analyzed with the FACScan flow cytometer, to confirm the efficiency of this transfection method. On Days 7, 14, 21 and 28 after the start of DC culture, DC were harvested and electroporated with M1 mRNA. The transfected DC were co-cultured with autologous UC blood CD34- cells. One week after the fourth priming of autologous CD34 negative cells with M1 mRNA electroporated DC, Ag-specific CTL activity was evaluated. To prepare target cells, M1 mRNA was added to autologous DC 48 h prior to CTL assays. RESULTS: Our CTL assays results indicate that UC blood CD34+ cell-derived DC transfected with M1 mRNA by electroporation stimulated Ag-specific CTL responses that are capable of recognizing and lysing autologous DC loaded with M1 mRNA. M1 mRNA transfected DC-primed CTL showed a significant cytotoxic activity against M1 mRNA loaded autologous DC, while nearly baseline cytotoxic activity was recorded for the M1 mRNA unloaded DC. DISCUSSION: Our results showed that mRNA-transfected DC are potent stimulators of T-cell immunity in vitro. In addition, mRNA-loaded DC can function as targets in CTL cytotoxicity assays, which offer a practical substitute for tumor cells in assays to test the immunological effects of specific Ags.

Cord Blood Stem Cell Transplantation↗
PubMed1993

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture.

In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC. No heterologous GJIC was observed between Cx26- and Cx43-transfected TMEC suggesting that heterotypic GJs do not form or that Cx26/Cx43 channels do not permit dye transfer.

Blotting, Western↗