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At least 37 records · Page 2Linked to original sources
PubMed1995

[Antitumor effect of interleukin-2 gene-transfected tumor vaccine in combination with interleukin-6 gene-transfected tumor vaccine].

It has been shown that IL-2 and IL-6 can cooperate to induce immune responses more effectively. In order to increase the therapeutic efficacy of cytokine gene-transfected tumor vaccines, we treated the experimental pulmonary metastatic melanoma-bearing mice with IL-2 gene-transfected tumor vaccine and IL-6 gene-transfected tumor vaccine. After the combined treatment, the pulmonary metastases were reduced more significantly and the survival time of tumor-bearing mice was also prolonged more significantly. The CTL activity, NK activity and IL-2-induced LAK activity, IL-2 and TNF secretion from the splenocytes of the above tumor-bearing mice increased more significantly than that of tumor-bearing mice treated with IL-2 gene-transfected vaccine or IL-6 gene-transfected vaccine alone. These results demonstrated that the combined use of IL-2 gene-transfected tumor vaccine and IL-6 gene-transfected tumor vaccine could achieve more potent antitumor effect via more efficient activation of immune functions. The experiment outlines a novel approach to the cytokine gene therapy of cancer.

Animals↗
PubMed1975

Effect of lysogeny on transfection and transfection enhancement in Bacillus subtilis.

Strains of Bacillus subtilis 168 lysogenic for bacteriophage phi105 transfer with deoxyribonucleic acid (DNA) isolated from bacteriophage SPO2 at a higher efficiency than non-lysogenic strains. This enhancement of transfection was not the result of recombination between bacteriophages SPO2 and phi105. Superinfection marker rescue increased transfection with DNA from bacteriophage phi105 occurred simultaneously with the addition of the transfecting DNA. Again, this enhancement of transfection was not the result of recombination but rather a protection of the transfecting DNA by the superinfecting bacteriophage. The ability of the superinfecting bacteriophage to protect the transfecting DNA from inactivation was maximal when the bacteria were just becoming competent. Bacteriophage phi1 cannot replicate after the transfection of competent bacteria lacking a functional DNA replication system, whereas bacteriophage phi1 was able to replicate after infection of competent bacteria grown under comparable conditions. These observations support the hypothesis that GAPase and an inducible repair system play an important role in the development of competence.

Azo Compounds↗
PubMed1975

Transfection of Escherichia coli spheroplasts. VI. Transfection of nonpermissive spheroplasts by T5 and BF23 bacteriophage DNA carrying amber mutations in DNA transfer genes.

DNA was extracted from T5 and BF23 phage carrying amber mutations in genes A2, A1, or D9 and tested for its ability to transfect su minus spheroplasts. DNA from T5 am231, defective in gene A2, transfects Escherichia coli su minus recB minus spheroplasts with an efficiency of 16% of that of wild-type T5 DNA, whereas DNA from T5 am16d or BF23 am57, both defective in gene A1 or its equivalent, transfects E. coli su minus recB minus spheroplasts with an efficiency of 1.4% of that of wild-type T5 DNA, provided E. coli su+ bacteria is used as the indicator in all cases. More than 95% of the progeny from the am231, am16d, and am57 DNA that transfects su minus recB minus spheroplasts is still amber mutant. From these efficiencies of transfection we conclude that the product of gene A2 functions mainly in the mechanism of transfer of phage DNA to intact host cells, and that this function is not essential for transfection of spheroplasts. We also conclude that gene A1 controls functions in addition to DNA transfer, in agreement with previous studies which show that mutations in gene A1 have a pleiotropic effect. Apparently, the absence of these additional functions controlled by gene A1 leads to a high frequency of abortive infection. DNA from amber mutants defective in either gene A1 or A2 does not appreciably transfect su minus rec+ spheroplasts, indicating that the products of these two genes may both be needed to protect T5 DNA from the very active rec BC nuclease in spheroplasts.

Coliphages↗
PubMed1993

Transfection of DNA into isolated rat adipose cells by electroporation: evaluation of promoter activity in transfected adipose cells which are highly responsive to insulin after one day in culture.

Isolated adipose cells are among the most insulin responsive cells with respect to glucose transport and metabolism. However, molecular biological techniques such as transfection of DNA have heretofore not been applied successfully in these cells in primary culture. We report a method for transfection of DNA into rat adipose cells by electroporation. Six shocks at 800 V and 25 microF in a 0.4 cm gap cuvette results in efficient transfection. We compared the ability of five promoters to drive expression of a luciferase reporter gene in transfected adipose cells. After one day in culture, promoter activity ranged from no expression to a very high level of expression. These transfected, cultured cells also displayed a 10-fold increase in 3-O-methylglucose transport with maximal insulin stimulation. The ability to transfect DNA into adipose cells which remain insulin responsive after one day in primary culture may be helpful for understanding adipose cell-specific gene regulation and elucidating the molecular mechanisms of insulin action.

3-O-Methylglucose↗
PubMed1978

Transfection with replicating DNA from the temperate Bacillus bacteriophage phi 105 and with T4-ligase treated phi105 DNA: the importance in transfection of being longer than genome-length.

Replicating phage DNA extracted from Bacillus subtilis infected with phage phi 105 has a higher activity in transfection than mature DNA. By heteroduplex analysis it was shown that this DNA contains concatemeric molecules. Concatemers, constructed in vitro by treatment of mature DNA with T4-ligase also have an increased activity in transfection. DNA showing an increased activity in transfection does not have a requirement for more than one molecule per transfection event as is typically found for transfection with mature phi 105 DNA. An explanation is given for this difference suggesting that the structure of the ends of the transfecting molecules play an important role intransfection.

Bacillus subtilis↗
PubMed2000

Zeta potential of transfection complexes formed in serum-free medium can predict in vitro gene transfer efficiency of transfection reagent.

We have tested the zeta potential (zeta, the surface charge density) of transfection complexes formed in serum-free medium as a rapid and reliable technique for screening transfection efficiency of a new reagent or formulation. The complexes of CAT plasmid DNA (1 microgram) and DC-chol/DOPE liposomes (3-20 nmol) were largely negatively charged (zeta=-15 to -21 mV), which became neutral or positive as 0.5 microgram or a higher amount of poly-L-lysine (PLL, MW 29300 or MW 204000) was added (-3.16+/-3.47 to +6.04+/-2.23 mV). However, the complexes of CAT plasmid DNA (1 microgram) and PLL MW 29300 (0.5 microgram or higher) were neutral or positively charged (-3.22+/-2.3 to +6.55+/-0.64 mV), which remained the same as 6.6 nmol of the liposomes was added. The complexes formed between two positively charged compounds, PLL MW 29300 (0.5 microgram) and the liposomes (3-20 nmol), were as closely positively charged as DNA/PLL or DNA/liposomes/PLL complexes (+3.31+/-0.41 to 7.16+/-1.0 mV). These results indicate that PLL determined the overall charge of the DNA/liposome/PLL ternary complexes. The complexes formed with histone (0.75 microgram or higher) were also positively charged, whose transfection activity was as high as PLL MW 29300. However, the complexes formed with protamine or PLL MW 2400 remained negatively charged. These observations are in good agreement with the transfection activity of the formulation containing each polycationic polymer. The presence of PLL MW 29300 did not change the hydrodynamic diameter of DNA/liposome/PLL complexes (d(H)=275-312 nm). The complexes made of different sizes of PLL (MW 2400 and 204000) also did not significantly change their size. This suggests that DNA condensation may not be critical. Therefore, zeta of the transfection complex can predict the transfection efficiency of a new formulation or reagent.

Animals↗
PubMed2004

Combined transfection with EBV-specific epitopes and HLA-A2 genes is more effective than separate transfection in promoting CTL lysis against nasopharyngeal carcinoma.

To augment specific cytotoxic T lymphocyte (CTL) lysis is a promising strategy for cancer therapy. In this study, we examined the boosting effect of CTLs upon autologous lymphoblastoid B cell lines (LCLs) transfected with diverse plasmids, to explore the possible CTL-based immunotherapy of nasopharyngeal carcinoma (NPC). FCM analysis displayed rather high ratio (>30%) of successfully transfected LCLs by utilizing the DMRIE-C kit. CTL assays demonstrated that substantially higher ratio of CTL specific lysis was observed upon the LCLs transfected with both expression vectors encoding EBV-specific epitopes and their presentation molecule HLA-A2, in contrast with those transfected separately. By transfecting the vector encoding HLA-A2 alone, only the LCLs of HLA-A2(+) donors elicited markedly higher CTL lysis. CTL assays also showed that there existed no marked differences upon transfection by either different vectors (pcDNA3, pNGVL3 or pNGVL3-hFlex), or different EBV-derived peptides (LMP(2)Pep1 or LMP(2)Pep2), or with or without the doubled DNA sequence encoding peptides. This study indicated a promising immunotherapy strategy on NPC through boosting and eliciting the EBV-specific CTL activation by transferring vectors encoding both EBV-specific epitopes and their presentation molecule HLA-A2 into autologous LCL, the presentation cells of MHC/peptide tetrameric complex.

Adult↗
PubMed1995

Overexpression of ribonucleotide reductase in transfected human KB cells increases their resistance to hydroxyurea: M2 but not M1 is sufficient to increase resistance to hydroxyurea in transfected cells.

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis. The enzyme consists of two subunits, M1 and M2. Hydroxyurea (HU) is an M2-specific inhibitor. It has been shown that a HU-resistant clone derived from stepwise exposure to HU overexpresses the M2 mRNA and the RR protein (Y. Yen et al., Cancer Res., 54: 3868-3691, 1994). In this study, we established stable clones by transfecting human KB cells with the cDNA of human wild-type RR in which each subunit was overexpressed by a SV40 promoter. The mammalian cell expression vector ph beta APr-1 was used for constructing M1, M2, and M1/M2 subunit cDNA. The transfected cells were selected with G418. The clones designated M2-D, M1-D, X-D, and KB-V represent transfectant clones which contain M2 cDNA, M1 cDNA, M1/M2 cDNA, and vector alone, respectively. The parental KB cells and clones containing vector plasmid KB-V express equally low amounts of M2 and M1 mRNA from the endogenous genes. The expression of M2 mRNA and M1 mRNA is elevated 2-3 fold in the X-D transfectants. M2-D clone demonstrated a 6-fold higher M2 mRNA level although the M1 mRNA expression remains the same as parental cells. M1-D transfectants have a 3-fold increase in M1 mRNA expression relative to parental cells, but reveal no alteration of M2 mRNA. Southern analysis of genomic DNA suggested the incorporation of the plasmid into the genome. The X-D clone revealed both integration of the M2 and M1 gene while the M2-D clone only showed M2 gene integration. The M1-D clone revealed M1 gene integration relative to the parental cells. The Western blot of M2 protein showed a 3-fold increase in the X-D and M2-D clones whereas the M2 protein level in M1-D was the same as it was in parental cells. The M1 protein was increased 3-fold in X-D and 1.5-fold in M1-D over that of parental cells. However, lower M1 protein levels were identified in the M2-D clone. The specific activity of the RR enzyme from each transfectant showed a 3-fold increase in both the X-D and M2-D clones and slightly increased in M1-D clone over that of parental cells. However, X-D and M2-D both demonstrated a 3-fold increase in resistance to HU as compared to M1-D which showed the same sensitivity as the parental enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Blotting, Southern↗
PubMed1998

Interaction of lipopolysaccharide with a mammalian lyso-phosphatidate acyltransferase (LPAAT) transfected into E. coli, and effect of lisofylline on LPAAT transfected into mammalian cells.

1. Lipid A and LPS stimulate LPAAT activity (and hence unsaturated PA formation) in RMC membranes and whole cells. 2. This correlates with cell phenotypic and membrane changes associated with small G proteins. 3. Unsaturated PA and Lipid A have similar effects on cells when given exogenously. 4. Human LPAAT-alpha and -beta isoforms were cloned and transfected into E. coli, demonstrating the ability to restore PA synthesis and reduce lyso-PA accumulation in plsC strains (LPAAT deficient mutants), as well as restoring growth at high temperatures. 5. LPAAT transfection into E. coli plsC (JC201) strains results in an increase in LPS content, suggesting stimulation of LPS synthesis. 6. LPAAT transfection into human A549 lung epithelial carcinoma and endothelial ECV304 cells results in increased cytokine mRNA transcription at baseline, and a significant increase in stimulated cytokine mRNA transcription. In addition, LPAAT transfection also results in increased cytokine release in response to IL-1 beta. 7. LSF, which reduces rodent deaths in sepsis models, reduces unsaturated acyl incorporation into PA in monoblastic cell lines, and reduces serum FFA increase in human sepsis, also reduces unsaturated acyl incorporation into PA in ECV304 cells. LPAAT-alpha transfection increases linolenate incorporation into PA at the expense of linoleate incorporation, which is reversed by LSF. LPAAT-beta increases both linoleate and linolenate incorporation into PA, which is also reduced by LSF. We conclude that LPAAT and PA remodeling may play a role in diffuse renal toxicity in sepsis due to induction of cellular phenotype changes associated with PA induction by Lipid A and/or LPS. Two human isoforms of LPAAT have been cloned, and apparently address C18 unsaturated acyl chains somewhat selectively. LSF causes functional reduction in LPAAT activity in transfected systems. This does not yet imply a direct effect of LSF on LPAAT. LPAAT and LPS may interact in the membrane in a not-yet-understood manner.

Acyltransferases↗
PubMed2003

Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency.

Large-scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins (r-proteins). As many r-proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purification is required. In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphate (SEAP) as reporter genes. The suspension growing, serum-free adapted HEK293SF-3F6 cell line was stably transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bearing plasmids. Compared to our standard serum-free medium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection efficiency and significantly increased volumetric productivity fourfold. Using the optimized medium formulation, transfection efficiencies between 40-60% were routinely obtained and SEAP production reached 18 mg/L(-1). To date, we have successfully produced and purified over fifteen r-proteins from 1-14-L bioreactors using this serum-free system. As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropilin-1 extracellular domains (ED) in bioreactors. Each protein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step. In contrast, purification of Tie-2 and Neuropilin-1 produced in serum-containing medium was much less efficient. Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free medium significantly improves protein expression compared to peptone-less medium, and significantly increases their purification efficiency compared to serum-containing medium. This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milligram amounts of r-proteins within 5 days posttransfection.

Bioreactors↗
PubMed2006

Infection, transfection, and co-transfection of baculoviruses by microprojectile bombardment of larvae.

The use of baculoviruses as expression vectors for heterologous proteins has been practically limited to the use of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In this work, infection, transfection and co-transfection events with the baculoviruses AcMNPV and Trichoplusia ni granulovirus (TnGV) were accomplished by bombardment of T. ni first-instar larvae with microprojectiles coated with virions, viral DNA, and viral DNA and a transfer vector, respectively. A series of shooting conditions were tested until positive results were obtained. The use of 1.6 microm gold particles at 900 psi shooting pressure, 400 Torr vacuum, 7 cm distance to target, on sets of 20 first-instar larvae held in a 16 mm diameter container, proved to be the best shooting conditions. Typical infection symptoms were shown by larvae when shot with viruses or viral DNA from AcMNPV or TnGV. Co-transfected recombinant AcMNPV and TnGV were identified by the formation of occlusion bodies and GFP, respectively, in bombarded larvae. This technique opens a wide range of possibilities, not only to use an extensive number of baculoviruses as expression vectors for heterologous proteins, but also be used to infect, transfect or co-transfect a wide variety of viruses into animal cells.

Animals↗
PubMed1998

A new efficient method for transfection of neonatal cardiomyocytes using histone H1 in combination with DOSPER liposomal transfection reagent.

Although cationic lipids are successfully used for gene transfer in vitro, primary cells such as neonatal cardiomyocytes frequently resist efficient transfection. We show here that the polycationic lipid DOSPER in combination with histone H1 was much more efficient in transfection of neonatal cardiomyocytes than DOSPER alone or other cationic lipids. This has been shown for transfection with the reporter plasmids pSV beta-gal and pCMV luc. If viral transfections are not possible, this mild method is an alternative to transfect cardiomyocytes.

Animals↗
PubMed1997

Co-transfection with protein kinase D confers phorbol-ester-mediated inhibition on glucagon-stimulated cAMP accumulation in COS cells transfected to overexpress glucagon receptors.

Glucagon elicited a profound increase in the intracellular cAMP concentration of COS-7 cells which had been transiently transfected with a cDNA encoding the rat glucagon receptor and under conditions where cAMP phosphodiesterase activity was fully inhibited. This was achieved in a dose-dependent fashion with an EC50 of 1.8+/-0.4 nM glucagon. In contrast with previous observations made using hepatocytes [Heyworth, Whetton, Kinsella and Houslay (1984) FEBS Lett. 170, 38-42], treatment of transfected COS-7 cells with PMA did not inhibit the ability of glucagon to increase intracellular cAMP levels. PMA-mediated inhibition was not conferred by treatment with okadaic acid, nor by co-transfecting cells with cDNAs encoding various protein kinase C isoforms (PKC-alpha, PKC-betaII and PKC-epsilon) or with the PMA-activated G-protein-receptor kinases GRK2 and GRK3. In contrast, PMA induced the marked inhibition of glucagon-stimulated cAMP production in COS-7 cells that had been co-transfected with a cDNA encoding protein kinase D (PKD). Such inhibition was not due to an action on the catalytic unit of adenylate cyclase, as forskolin-stimulated cAMP production was unchanged by PMA treatment of COS cells that had been co-transfected with both the glucagon receptor and PKD. PKD transcripts were detected in RNA isolated from hepatocytes but not from COS-7 cells. Transcripts for GRK2 were present in hepatocytes but not in COS cells, whereas transcripts for GRK3 were not found in either cell type. It is suggested that PKD may play a role in the regulation of glucagon-stimulated adenylate cyclase.

Animals↗
PubMed1997

An in vitro 1H-MRS model of oncogene transfection. The spectral feature of c-erbB-2 and c-Ha-ras transfected NIH3T3 fibroblast cells.

PURPOSE: Malignancy is an abnormality of cell division and differentiation based on abnormal expression of oncogenes. This note describes the in vitro 1H-NMR spectral features of oncogene-transfected NIH3T3 fibroblast cells compared to non-transfected cells. MATERIAL AND METHODS: 1H-NMR spectra of cultured NIH3T3 cells and c-erbB-2 or c-Ha-ras gene-transfected cells were obtained by 400 MHz high resolution NMR. The peaks were assigned by 2D HOHAHA spectra of the cell suspension and the spectral changes were evaluated in 1D and 1D differential spectra. RESULTS: The 1H spectra obtained from both transfected cell lines were broadened over all peaks, suggesting reduced mobility in plasma membrane lipid molecules. No other differential spectra for characterizing metabolic change was detected. CONCLUSION: Broadened 1H spectra observed after c-erbB-2 or c-Ha-ras transfection suggest changes of plasma membrane viscosity, which may be related to the oncogene expression.

3T3 Cells↗
PubMed1995

Transient transfection of GGH3-1' cells [GH3 cells stably transfected with the gonadotropin-releasing hormone (GnRH) receptor complementary deoxyribonucleic acid] with the carboxyl-terminal of beta-adrenergic receptor kinase 1 blocks prolactin release: evidence for a role of the G protein beta gamma-subunit complex in GnRH signal transduction.

G proteins consist of heterotrimeric alpha-, beta-, and gamma-subunits. To assess the role of the beta gamma-subunit complex in GnRH receptor-mediated signal transduction, GGH3-1' cells were transfected with plasmids PRK5-beta ARK1 (495-689) containing complementary DNA (cDNA) of the carboxyl-terminal (Gly495-Leu689) of beta-adrenergic receptor kinase 1 (beta ARK1). GGH3-1' cells are GH3 cells that have been stably transfected with rat GnRH receptor cDNA. The carboxyl region of beta ARK1 (Gly495-Leu689) binds to the beta gamma complex and thereby inhibits its action. Twenty-four hours after stimulation, PRL release, cAMP release, and inositol phosphate (IP) production were measured in these cells and in control cells transfected with vector PRK5 cDNA alone. In cells expressing the beta ARK1-(495-689) sequence there was inhibition of basal PRL release (69.3%), cAMP release (61.2%), and IP production (75.5%) compared to cells containing vector only. When cells expressing the beta ARK1 fragment were stimulated with a GnRH analog (Buserelin; 10(-7) M), maximal responses were inhibited (66.1% for PRL release, 52.3% for cAMP release, and 79.1% for IP production). Scatchard analysis of GnRH analog binding was also performed in the two groups of transfected cells. No significant differences in Kd or receptor numbers were found between beta ARK1-(495-689)-transfected cells and control cells containing the vector alone. These data suggest a role for the beta gamma complex in mediation of cAMP release, IP production, and hormone release in response to agonist occupancy of the GnRH receptor.

Animals↗
PubMed2004

[In vitro gene transfection by magnetic iron oxide nanoparticles and magnetic field increases transfection efficiency].

OBJECTIVE: To evaluate the feasibility of using iron oxide nanoparticles as gene vector and the effect of magnetic field on efficiency of transfection. METHODS: Iron oxide nanoparticles were prepared by alkaline precipitation of divalent and trivalent iron chloride. The surface of iron oxide nanoparticles was modified by self-assembled poly-L-lysine to form particle complexes (IONP-PLL). Transfection was determined by delivering reporter gene, PGL2-control encoding luciferase, to different cell lines using IONP-PLL as vector. The effect of magnetic field on efficiency of transfection was determined using Nd-Fe-B permanent magnet. RESULTS: Foreign gene could be delivered to various cell lines by IONP-PLL and expressed with high efficiency, but the transfection efficiency and time course varied in the different cell lines studied. Magnetic field could enhance the efficiency of transfection by 5 - 10 fold. CONCLUSION: IONP-PLL can be used as a novel non-viral gene vector in vitro, which offers a basis for gene delivery in vivo.

Animals↗