lipofectamine and calcium-phosphate--monobasic--anhydrous

To optimise the high efficiency, non-viral transfer of DNA to retinal pigment epithelial (RPE) cells in vitro.. A mammalian expression vector (pcDNA3.1) containing a firefly luciferase (luc) cDNA was used to transfect RPE cells using different chemical methods; calcium phosphate, DEAE-dextran and, liposomes-based transfection techniques. Transfection was optimised for both dose and time of exposure. The efficiency of gene transfer and cytotoxicity was measured 48 hours post-transfection using luciferase and MTT assays, respectively. The percentage of transfected cells (using optimal conditions) was determined with a construct expressing a jellyfish green fluorescent protein (GFP) using flow cytometery.. Calcium phosphate and DEAE-dextran techniques failed to transfect the vector and led to high cytotoxicity. Liposomes-based methods successfully transferred the vector to RPE cells, but the efficiency varied for different liposomes; Tfx-50 > Lipofectin > Lipofectamine > Cellfectin > DMRIE-C. No significant cytotoxicity was observed with any of the liposome treatments. Optimal transfection was achieved with Tfx-50 at a 3:1 ratio of DNA:liposome; between 12-15% of cells being transfected.. Efficient and non-toxic transfer of functional genes into primary RPE cells in vitro can be successfuly achieved by liposomes-based techniques. Tfx-50 appears to be a promising non-viral vector for RPE gene transfer.

Topics: Aged; Calcium Phosphates; Cation Exchange Resins; DEAE-Dextran; DNA; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression; Genetic Vectors; Green Fluorescent Proteins; Humans; Lipids; Liposomes; Luciferases; Luminescent Proteins; Middle Aged; Phosphatidylethanolamines; Pigment Epithelium of Eye; Quaternary Ammonium Compounds; Time Factors; Transfection

Polylysine (pLy) has been used as a DNA carrier in nonviral gene delivery systems because it forms complexes with plasmid DNA via charge interaction, and condenses it into a compact structure. We have recently shown that cross-linking nuclear localization sequences (NLSs) to pLy can enhance transfection by conferring specific recognition by the cellular nuclear import 'receptor', the NLS-binding importin alpha/beta heterodimer. The present study examines and correlates for the first time the effect of the lysine/nucleotide (Ly/Nu) ratio on transfection, recognition by importin alpha/beta, and structure as determined using electron microscopy (EM) and atomic force microscopy (AFM), for pLy-DNA complexes with and without NLSs or mutant versions thereof. Intriguingly, we observed two distinct peaks of transfection enhancement at Ly/Nu ratios of 0.4 and 4.0, attributable to specific NLS recognition by importins and DNA compaction, respectively. The results indicate a clear correlation between the pLy-DNA structure, importin alpha/beta recognition, and gene transfer efficiency, thus underlining the importance of using pLy-DNA at the optimal Ly/Nu ratio.

Topics: Animals; beta-Galactosidase; Calcium Phosphates; Cation Exchange Resins; Drug Resistance, Microbial; Genetic Therapy; Genetic Vectors; Karyopherins; Lipids; Luminescent Measurements; Microscopy, Atomic Force; Microscopy, Electron; Neomycin; Nuclear Localization Signals; Nuclear Proteins; Plasmids; Polylysine; Rats; Transfection; Tumor Cells, Cultured

To identify a good system to introduce foreign genes into normal and tumoral astrocytes, we studied the efficiency of two chemical methods, calcium phosphate precipitation and lipofection, and of a viral-mediated transfer by a vector derived from the highly attenuated modified vaccinia virus Ankara (MVA). Using the beta-galactosidase (beta-gal) gene (lacZ) as reporter, we searched for optimal experimental conditions to obtain an efficient gene transfer into human embryonic and neonatal rat astrocytes and into a human astrocytoma cell line (U373 MG). The beta-gal protein production was evaluated by cytochemical staining and enzymatic activity assay. Among chemical methods, lipofection was the most efficient system to transfect astrocytes in providing up to 60% of beta-gal-positive cells in all the cell types analyzed. MVA infection also proved to be an efficient system to introduce heterologous genes into human embryonic astrocytes that appeared 80-100% positive 48-96 hr after an infection at a multiplicity of 1-10. In contrast, only a limited infection was observed with rat astrocytes, human astrocytoma cells, and human leptomeningeal cells. A recombinant MVA vector expressing the human immunodeficiency virus-1 (HIV-1) regulatory protein Nef was used to transfect human embryonic astrocytes, and the resulting Nef expression was compared with that detected after lipofection in the same cells. By Western blot analysis, Nef expression was observed in human astrocytes 24-96 hr after infection and was similar to that present in stably HIV-1-infected astrocytoma cells. Lipofection resulted in lower Nef expression. In spite of these promising results, the negative effects of MVA infection on cell viability and the possibility that a productive infection occurs in human embryonic astrocytes limit the use of this vector for gene delivery in developmentally immature human glial cells.

Topics: Animals; Astrocytes; Calcium Phosphates; Cation Exchange Resins; Cell Size; Cell Survival; Cells, Cultured; Chemical Precipitation; Gene Expression; Gene Products, nef; Gene Transfer Techniques; Genetic Vectors; HIV-1; Humans; Lipid Metabolism; Lipids; nef Gene Products, Human Immunodeficiency Virus; Rats; Recombinant Proteins; Time Factors; Transfection; Tumor Cells, Cultured; Vaccinia virus

mRNA for the regulatory subunit RIIbeta of cAMP-dependent protein kinase is stimulated more than 50-fold by cAMP in primary cultures of rat Sertoli cells. We have previously shown that this induction involves regulation of transcriptional activation as well as mRNA stabilization. The rat RIIbeta gene contains no cAMP response element (CRE), and the induction of RIIbeta mRNA is slow and requires on-going protein synthesis. When a construct containing the 5'-flanking region of the RIIbeta gene upstream of a CAT reporter was transfected into Sertoli cells by the calcium phosphate method, low and variable responses to cAMP (three- to fivefold) were observed, whereas a 15- to 20-fold increase in reporter activity by cAMP was observed after lipofectamine transfection. Interestingly, when a vector containing CRE elements upstream of a reporter gene was transfected into Sertoli cells, the responses to cAMP were similar regardless of the transfection method used. We have also demonstrated that increased intracellular levels of calcium by A23187 and thapsigargin dramatically inhibit cAMP-mediated induction of RIIbeta mRNA, but not the mRNA for the CRE-containing RIalpha gene. Furthermore, decreased cAMP responsiveness of endogenous RIIbetamRNA (but not RIalpha) was also observed in calcium phosphate-transfected Sertoli cells but not in lipofectamine-transfected cells. Thus, calcium-mediated reduction in cAMP response appears to be a gene-specific phenomenon.

Topics: Animals; Calcimycin; Calcium; Calcium Phosphates; Cation Exchange Resins; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Induction; Enzyme Inhibitors; Ionophores; Lipids; Male; Rats; Rats, Sprague-Dawley; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Thapsigargin; Transfection

Monocyte/macrophage cell lines are fastidious cells commonly used in transient transfection experiments. In the course of a study of gene regulation by lipopolysaccharide (LPS), we have compared several methods for DNA-mediated cell transfection to determine which would be optimally applicable to the macrophage line, RAW 264.7. Both the response level (LPS inducibility) and the degree of inter-assay variation were evaluated for each transfection technique. The following methods were compared: Lipofectin, LipofectAMINE, LipofectAMINE PLUS, SuperFect, Ca3(PO4)2 DNA co-precipitation, DEAE dextran-mediated transfection and electroporation. The transfected plasmid DNA included a luciferase reporter construct containing the junB minimal promoter under the control of an LPS-inducible 1300-bp regulatory fragment downstream of junB 5'-flanking sequence, as well as a beta-galactosidase reporter construct under the adenovirus promoter and enhancer used as an internal control. Electroporation, followed by a resting period of 16-24 h before stimulation with LPS, had the highest inducibility of all methods. DEAE dextran and Ca3(PO4)2 precipitation showed the least and the greatest inter-assay variation, respectively. For all other methods, inter-assay variability fell within this range. The results presented may serve as both a general reference and a guide for reporter gene studies in this or other macrophage cell lines.

Topics: Adenoviridae; Animals; beta-Galactosidase; Calcium Phosphates; Cation Exchange Resins; Cell Line; Chemical Precipitation; DEAE-Dextran; DNA; Electroporation; Genes, jun; Genes, Reporter; Lipids; Lipopolysaccharides; Luciferases; Macrophages; Mice; Phosphatidylethanolamines; Plasmids; Promoter Regions, Genetic; Transfection

The establishment of gene delivery systems that result in efficient transfection of the pancreatic beta-cells may generate an important tool for the study of IDDM and may also represent one critical step toward a clinical application of gene transfer for the prevention or early treatment of the disease. Using the reporter gene vectors pCAT and pCMV beta-gal, we have investigated the efficiency of transfection mediated by calcium phosphate precipitation, the monocationic liposome Lipofectin, the polycationic liposome Lipofectamine, and adenovirus-polylysine (AdpL) DNA complexes in human, mouse, rat, and fetal porcine islet cells. In all species studied, calcium phosphate-mediated transfection resulted in lower chloramphenicol acetyl transferase (CAT) activities than the other methods. Intact human, mouse, and rat islets were poorly transfected by Lipofectin, Lipofectamine, and AdpL. When dispersed by trypsin treatment, however, human, mouse, rat, and fetal pig islect cells were efficiently transfected by Lipofectamine. Moreover, transfection of dispersed human and mouse islet cells using AdpL, also resulted in high CAT activities. The percentage of cells staining positively for beta-galactosidase after transfection with Lipofectamine was 49% for mouse, 56% for rat, and 57% for dispersed human islet cells. Transfection of human islet cells using AdpL, however, yielded 70% beta-gal-positive cells. Fluorescence-activated cell sorting-purified rat islet alpha- and beta-cells were transfected with similar efficiency using Lipofectamine. CAT expression in human islet cells transfected with either Lipofectamine or AdpL reached a peak value after 5-7 days, followed by a gradual decline. It is concluded that transfection with AdpL or Lipofectamine are both efficient means to achieve transient expression of gene constructs in human and mouse islet cells, while for rat and fetal porcine islet cells, Lipofectamine is the most efficient of the agents investigated in this study.

Topics: Analysis of Variance; Animals; beta-Galactosidase; Calcium Phosphates; Cation Exchange Resins; Cell Survival; Cells, Cultured; Chloramphenicol O-Acetyltransferase; Cytomegalovirus; Fetus; Genes, Reporter; Genetic Vectors; Humans; In Vitro Techniques; Indicators and Reagents; Islets of Langerhans; Lipids; Liposomes; Mice; Phosphatidylethanolamines; Polylysine; Rats; Swine; Transfection

Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and therefore are major target cells for different aspects of gene transfer. Previous attempts to transfect primary arterial cells using different techniques like liposomes, CaPO4 and electroporation resulted in only low transfection efficiency. The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cell types including SMC. However, cloning and identification of recombinants remain difficult and time-consuming techniques. The present study demonstrates that a complex consisting of reporter plasmid encoding firefly luciferase (pLUC), polycationic liposomes and replication-deficient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions. The technique of adenovirus-assisted lipofection (AAL) increases transfer and expression of plasmid DNA in human smooth muscle cells in vitro up to 1000-fold compared to lipofection. To verify the applicability of AAL for gene transfer into human smooth muscle cells we studied a gene therapy approach to suppress proliferation of SMC in vitro, using the prokaryotic cytosine deaminase gene (CD) which enables transfected mammalian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-fluorouracil (5-FU). The effect of a transient CD expression on RNA synthesis was investigated by means of a cotransfection with a RSV-CD expression plasmid and the luciferase reporter plasmid. Western blot analysis demonstrated high expression of CD protein in transfected SMC. Cotransfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected with a non-CD coding plasmid. The data demonstrate that a transient expression of CD could be sufficient to reduce the capacity of protein synthesis in human SMC. This simple and effective in vitro transfection method may also be applicable to in vivo delivery of target genes to the vascular wall to inhibit SMC proliferation.

Topics: Adenoviruses, Human; Aorta; Calcium Phosphates; Cation Exchange Resins; Cells, Cultured; Cytosine Deaminase; Defective Viruses; DNA, Recombinant; Gene Transfer Techniques; Genes, Reporter; Genetic Vectors; Humans; Lipids; Liposomes; Luciferases; Muscle, Smooth, Vascular; Nucleoside Deaminases; Recombinant Fusion Proteins; RNA; Transfection