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PubMed1995

Rescue of injury-induced neurofilament loss by BDNF gene transfection in primary septo-hippocampal cell cultures.

We employed primary septo-hippocampal cell cultures to determine the ability of liposome-mediated BDNF gene transfection to facilitate recovery of neurofilament loss caused by depolarization injury. After BDNF gene transfection in uninjured cultures, RT-PCR and immunohistochemical staining confirmed increases in BDNF mRNA and protein in transfected cells. Three days after depolarization injury, Western blot and immunohistochemical analyses detected significant loss of neurofilament proteins in non-transfected cultures, while BDNF transfection produced marked increases in neurofilament proteins following either pre-injury transfection or transfection 24 h following injury. Immunohistochemical studies also detected enhanced immunolabeling of BDNF and total neurofilament protein (phosphorylated and non-phosphorylated) in injured neurons following BDNF transfection or administration of exogenous BDNF protein, compared to untransfected, injured controls.

Animals↗
PubMed1988

Transfection of the hypotrichous ciliate Stylonychia lemnae with linear DNA vectors.

We present a new protocol for the transfection of Stylonychia lemnae with linear DNA vectors containing the neomycin-resistance gene from Escherichia coli transposon Tn5. The taking up of heterologous DNA is achieved by damaging the cells' protein coat with urea prior to transfection according to the calcium phosphate co-precipitation procedure. After transfection, transformed cells can be enriched by selection with the antibiotic drug G418. Hybridization experiments show that macronuclear DNA of these G418-selected cells contains molecules homologous to the transfected vector DNA, which are altered by some recombination process. Transfected cells, which have grown for more than 100 cell cycles in antibiotic-free medium, still contain vector-homologous DNA, but recombination continued during this time. We are able to transfect Stylonychia early after conjugation, a process which is followed by complex genome rearrangements and amplification. In these experiments we observed considerable amplification of vector-homologous DNA molecules as compared to transfected vegetative cells. Again these molecules are altered by recombination in respect to the original vector DNA. As soon as suitable vectors are available, the transfection protocol presented here can be a basic tool for the study of DNA replication, transcription and macronuclear development in Stylonychia.

Animals↗
PubMed1991

A novel method for generating stable high-level transfectants involving direct immunomagnetic selection for cell-surface epitopes: expression of HLA class-II genes in HeLa cells.

A versatile method that allows efficient detection and selection of both transient and stable transfectants expressing exogenous cell-surface molecules is described and used to generate stable HeLa transfectants expressing each of the human HLA class-II isotypes, specifically the DR1, DQw8 and DPw2 heterodimers. The method combines use of the strong mammalian expression vector, CDM8, and a highly efficient transfection protocol with the powerful technique of immunomagnetic selection. It offers significant advantages in comparison to standard procedures involving co-selection with drug-resistance markers. The transfection efficiency can be assessed 60 h after transfection rather than after three weeks of drug selection. Repeated rounds of immunomagnetic selection applied over the subsequent ten days result in homogeneous populations which express the surface marker of interest stably at high levels, making further subcloning or fluorescence-activated cell sorting unnecessary. Any number of surface products can be transfected into the same cell, the only limitation being the availability of specific monoclonal antibodies (a DP/DR double transfectant is described expressing four exogenous gene products simultaneously). The high sensitivity of immunomagnetic selection and its applicability to large samples allows rescue of transfectants present at very low frequencies. Finally, the technique can be used as a coselection procedure, by analogy with drug coselection, to achieve expression even of non-cell surface products.

Cloning, Molecular↗
PubMed2005

Optimized transfection of mRNA transcribed from a d(A/T)100 tail-containing vector.

Studies employing mRNA transfection are currently limited by a lack of transcription vectors for generating a long poly(A) tail-containing mRNA and published methods for efficient mRNA transfection. We have constructed a transcription vector containing firefly luciferase gene (pBS-FLuc-A100) to generate luciferase mRNA with A100 tail followed by no heterologous sequence. The pBS-FLuc-A100 was propagated in XL1-Blue, in which the plasmid was more stable than in other bacterial strains. Optimal mRNA transfection conditions were determined using TransMessenger Transfection Reagent (Qiagen) and yeast tRNA as a carrier. Firefly luciferase expression, which peaked at about 12 h post-transfection, was detected with as little as 5 ng mRNA and was linear with mRNA amount up to 100 ng. When cells were transfected with luciferase mRNA containing different lengths of poly(A) tail, luciferase expression increased proportionally with poly(A) tail length up to 60A residues and then declined. Cell lines from monkey, mouse, and rat were transfected efficiently by this method. Like cellular ferritin heavy chain mRNA, which contains an iron response element in its 5'UTR, translation of transfected luciferase mRNA containing the 5'UTR of ferritin mRNA was iron-dependent. Our results demonstrate that the poly(A) vector and the transcription method described will be useful to study the regulation of gene expression at the mRNA level by UTRs.

Animals↗
PubMed2006

Cell-surface streptavidin fusion protein for rapid selection of transfected mammalian cells.

We developed a series of eight mammalian cell surface marker fusion genes by using the streptavidin gene from Streptomyces avidinii. These fusion genes are useful and non-growth-toxic selection markers for rapid-harvest transfected mammalian cells. Two streptavidin constructs were used; the longer fragment contains the native bacterial signal sequence, which the shorter fragment lacks. For expression of the streptavidin antigen on the surface of mammalian cells, streptavidin was flanked by a mammalian signal sequence and a transmembrane domain (from mouse H2-K or Kit); some constructs also contained the gene for enhanced green fluorescent protein (EGFP). We transfected a series of plasmids encoding the fusion proteins into HeLa cells and determined that the transfected cells produced the fusion protein on their cell surfaces. To separate transfected cells from nontransfected cells, we incubated cells with a polyclonal antibody against streptavidin, and antibody-bound cells were harvested by the use of paramagnetic beads coupled with the corresponding secondary antibody. We obtained highly pure populations of transfected cells; this result also confirmed the production of the fusion protein on the cell surface. Cell growth assays revealed that none of the transfected fusion genes or their products adversely affected the proliferation of HeLa cells. Our results indicate that the fusion constructs we developed and the immunomagnetic separation protocol we used are valuable tools for various transfection applications. In particular, the constructs containing EGFP are advantageous because transfection efficiency can be assessed without additional treatment of cells.

Cell Membrane↗
PubMed2006

pH dependent high transfection efficiency of mouse neuroblastomas using TransFectin.

Mouse neuroblastoma cell lines are often used in lieu of mouse primary neurons in ex vivo experiments, as they provide an easier platform for transfection, compared to the latter. A well-known inherent problem with this strategy is the relatively low transfection efficiency (15-30%) of mouse neuroblastoma cell lines such as neuro-2A and N1E-115. We were able to improve the transfection efficiency of these cell lines by using the cationic lipid reagent, TransFectin (Bio-Rad, Hercules, CA, USA) to optimise the transfection conditions. Our results, based on fluorescence intensity determinations and Western blotting for enhanced green fluorescence protein (EGFP) over-expression in neuro-2A, demonstrated that pH is a crucial factor in determining the transfection efficiency. Under pH-optimised transfection conditions, flow cytometric analysis revealed high EGFP transfection efficiencies of 76.4 +/- 0.5 and 60.9 +/- 0.6% for neuro-2A and N1E-115, respectively. Notably, the optimised TransFectin-based transfection system did not result in any detectable cytotoxicity to the mouse neuroblastomas. The resultant optimised system is economical, easy to use and does not require any specialised equipment.

Animals↗
PubMed2005

New trends in tumor biology: transfection of a human hepatoblastoma cell line with green fluorescent protein.

BACKGROUND/PURPOSE: Enhanced green fluorescent protein (eGFP) is widely used as a marker in different biologic systems. After vector transfection, eGFP is expressed by eukaryotic cells and can be visualized using fluorescent microscopy. The aim of this study was to establish an eGFP-transfected human hepatoblastoma (HB) cell line as tool for further basic research studies. METHODS: The HB cell line HUH6 was transfected with the pEGFP-N1 vector by liposomal transfection. Enhanced green fluorescent protein-positive cells were sorted out by fluorescence-activated cell sort and selected using G418 resistance. Expression of eGFP-messenger RNA was assessed by single-cell reverse transcriptase polymerase chain reaction after laser microdissection. Original and transfected cells were compared biologically and cytomorphologically. RESULTS: Vector transfection produced up to 15% eGFP-positive cells. After fluorescence-activated cell sort and G418 selection, a pure cell line was established with 100% eGFP-positive cells. Reverse transcriptase polymerase chain reaction revealed constant expression of eGFP-messenger RNA in fluorescending cells. Analysis of cell characteristics revealed no differences between transfected and original cells. CONCLUSIONS: For the first time, the authors established an eGFP-transfected HB cell line. This cell line can serve as a promising tool for further studies investigating HB in vitro and in vivo. Our model might also be a basis for similar work on other pediatric solid tumors.

Flow Cytometry↗
PubMed2006

Visualization of xenotransplanted human rhabdomyosarcoma after transfection with red fluorescent protein.

BACKGROUND/AIMS: Discosoma sp red fluorescent protein (DsRed2) is a newly developed marker for in vivo labeling studies in different biologic systems. After vector transfection, DsRed2 is expressed in mammalian cells and can be detected by fluorescence microscopy. The aims of this study were to establish a DsRed2-transfected human rhabdomyosarcoma (RMS) cell line and to perform a xenotransplantation on nude mice to use imaging as a tool for further basic research studies on this neoplasm. PROCEDURE: The human alveolar RMS cell line Rh30 was transfected with the pDsRed2-N1 vector by lipofection. The DsRed2-positive cells were sorted out by fluorescence-activated cell sorting analysis 96 hours after transfection and selected in culture with G418. Expression of DsRed2 messenger RNA was assessed using single-cell reverse transcriptase polymerase chain reaction after laser microdissection. Transfected and parental cells were characterized cytologically, cytogenetically, immunohistochemically, and in vivo after subcutaneous injection in NMRI (nu/nu) nude mice. RESULTS: After vector transfection, a pure and stable DsRed2-positive cell line was established by monoclonal growth of the cells. Reverse transcriptase polymerase chain reaction revealed constant expression of DsRed2 messenger RNA in fluorescencing cells. There was no difference between transfected and parental cells by means of cell morphology and desmin expression. Clonal cells (1 x 10(6)) were used for xenotransplantation. Tumors were visualized noninvasively through the skin of the mice using specific emission and excitation filters. Tumor vascularization and vessel growth could be discriminated from tumor tissue using this imaging system. CONCLUSION: This is the first report on successful transfection of an RMS cell line with red fluorescent protein followed by xenotransplantation into nude mice. This model can serve as an imaging tool for in vivo studies investigating tumor biology and metastases of human RMS.

Animals↗
PubMed2004

Effect of antisense VEGF cDNA transfection on the growth of chronic myeloid leukemia K562 cells in vitro and in nude mice.

To further elucidate the role of vascular endothelial growth factor (VEGF) in the pathogenesis of chronic myeloid leukemia (CML), we transfected K562 cells with a VEGF(121)cDNA sense vector (S), an antisense (AS) vector or vector (V) alone. The growth of transfected cells was investigated by MTT and colony-formation assays, and apoptosis was measured by flow cytometry (FCM) of Annexin-V-FITC/PI dual labeled cells. Transfected cells were subcutaneously transplanted into nude mice and the microvessel density (MVD) of tumor masses was determined by vWF immunohistochemistry staining. We tested the supernatant of different transfected K562 cells against human bone marrow endothelial cells (BMECs), and examined the synergic effects of antisense VEGF(121)cDNA and IFNalpha or STI571 on the proliferation and apoptosis of K562 cells. We found that K562/AS transfectants exhibited a 49% reduction in VEGF secretion, whereas K562/S transfectants exhibited a 3-fold increase in VEGF secretion, all in comparison to the vector controls. K562 cells transfected with antisense VEGF(121)cDNA showed growth retardation in vitro. In transplanted nude mice in vivo, transfection of implanted cells with antisense VEGF(121)cDNA resulted in decreased tumor MVD, and increased apoptosis in the presence of IFNalpha. Taken together, these results suggest that VEGF may be involved in the pathogenesis of CML through autocrine and paracrine mechanisms, and that anti-VEGF therapy alone or in combination with conventional treatment may be beneficial for CML patients.

Animals↗
PubMed2004

Heparin can improve the viability of transfected cystic fibrosis cell lines in vitro.

Cationic liposomes are widely used as gene transfer agents in in vitro and in vivo studies of cystic fibrosis. In this study we report comparative results of cationic mediated transfection in several cell lines. We have tested epithelial cell lines expressing the wild-type cystic fibrosis transmembrane protein CFTR (bronchial epithelium-16HBE14o-, submucosal gland-Calu3) and their cystic fibrosis counterparts (CFBE41o-, CFSMEo-), as well as baby hamster kidney fibroblast cell lines (BHK) heterologously expressing human CFTR. The cells were transfected with a green fluorescent protein plasmid complexed with commercial cationic liposome (Geneporter2, GP) and 25 kDa polyethylenimine (PEI). At the end of the incubation (2 hours), low molecular weight heparin was added in order to reduce the toxicity of the lipoplexes. Transfection efficiency and cell viability were measured by flow cytometry. Determination of fatty acid composition of cellular phospholipids was performed by capillary gas chromatography. The short incubation time was sufficient to obtain satisfactory transfection in all cell lines studied. Cells treated with PEI-complexes had lower transfection efficiency and viability compared to GP in all tested cell lines. DeltaF508 CFTR carrying airway epithelial cells were easier to transfect but had lower viability compared to their healthy counterparts. This was, however not the case for the BHK cells. The fatty acid analysis showed characteristic polyunsaturated fatty acid patterns, which correlated with the viability of the transfected cells. Low molecular mass heparin added at the end of the lipoplex incubation time could help to maintain the viability of the cells, without interfering with the transfection efficiency.

Animals↗
PubMed2006

Mouse CD40-transfected cell lines cannot exhibit the binding and RANTES-stimulating activity of exogenous heat shock protein 70.

Here we demonstrate the inducible mouse Hsp72 binds markedly to lymphoid neoplastic macrophage-like P388D1 cells. To examine whether mouse CD40 can play a role in signaling exogenously administered HSP70 in a fashion similar to that of human CD40, we established mouse CD40-transfectants of both human 293 cells and murine-pro-B cell line Ba/F3. A small portion of mouse CD40 expressed on 293-derived transfectants was the mature form with a signal-transducible C-terminal domain, whereas a majority of expressed antigen showed the molecular size smaller than we expect. Flow cytometry showed that mouse Hsp72, but neither its deletion variants nor the related Escherichia coli DnaK, bound to the 293-derived transfectants regardless of CD40 expression. CD40 molecules expressed on the transfectants showed the binding of soluble form of CD40L but this binding was not inhibited by excess amount of HSP70. CD40L, but not any HSP70 recombinant proteins, stimulated the production of chemokine RANTES in the transfectants. Furthermore, no RANTES production was induced by HSP70-RCMLA complex in the transfectants, although it binds to 293-derived cells in a CD40-independent manner. No interaction between mouse CD40 and HSP70 recombinant proteins was detected by using the Ba/F3-derived transfectants that express the mature form of mouse CD40. The present results imply that mouse CD40 expressed on the transfectants differs from its human homolog in the binding of exogenously administered HSP70.

Animals↗
PubMed2004

Enhanced effect of myocardial gene transfection by VP22-mediated intercellular protein transport.

One of the major issues in myocardial gene therapy is poor transfection efficiency. The herpes simplex virus protein VP22 is known to facilitate intercellular protein transport. Not only VP22 but also VP22-linked protein are exported from the cytoplasm of cells, in which it is synthesised endogenously, and transferred to surrounding cells, where it is translocated into the nuclei. However, the feasibility and efficiency of the intercellular trafficking properties of VP22-linked protein in the myocardium has not been clarified. Rat hearts were transfected by direct intramyocardial injection of naked plasmid vectors encoding either lacZ or VP22-linked lacZ. At day 5 following transfection, similar numbers of cardiomyocytes surrounding the injection sites showed beta-galactosidase (beta-gal) expression in the cytoplasm in both groups. In addition to this, following transfection of VP22-linked lacZ, most of the cardiomyocytes adjacent to the cytoplasmic-positive cells demonstrated nuclear-localised beta-gal expression. The number of these nuclear-positive cardiomyocytes, which are thought to be secondary protein-transported cells, was 4.3-fold greater than that of primary transfected, cytoplasmic-positive cells. Western blot analysis demonstrated that the amount of targeted protein expression is 2.9-fold greater following VP22-lacZ transfection (VP22-linked beta-gal; approximately 40 kDa bigger than wild-type beta-gal) compared with lacZ transfection (wild-type beta-gal). This data highlights the efficiency of the VP22-mediated intercellular protein delivery in the myocardium following in vivo gene transfection and suggests that the VP22-mediated effect is useful in enhancing the efficacy of myocardial gene therapy.

Animals↗
PubMed2004

FuGENE 6 Transfection Reagent: the gentle power.

FuGENE 6 Transfection Reagent has been commercially available since 1997. Since that time, its popularity has increased due to its ease of use, minimal to no cytotoxicity, and the high level of transfection in many different cell lines. FuGENE 6 Transfection Reagent is gentle on the cells. Adherent cells can be trypsinized and transfected by the DNA:FuGENE 6 reagent complex prior to plating, making it a strong candidate for high throughput applications. Additionally, low cell numbers can be transfected in 96-well plates. As with most reagents, the complex formation step is critical and special handling is required because the reagent is supplied in 80% ethanol. For example, contact with plastic must be avoided as inhibitors of transfection can leach from some plastics. We investigated parameters that have been reported to affect the transfection efficiency including the use of common antibiotics, passage level of the cells, and length of time for complex formation. These parameters are often cell line dependent and can be optimized to increase transfection efficiency for a specific cell line.

Animals↗
PubMed1997

Gene transfer mediated by cationic lipids: lack of a correlation between lipid mixing and transfection.

Complexes of DNA with cationic lipids are used to transfect eukaryotic cells. The mechanism of transfection is unknown, but it has been suggested that the complexes are taken up into the cell by endocytosis, after which fusion of the cationic lipids with the membranes of intracellular vesicles would allow the DNA to escape into the cytoplasm. Here, we have compared transfection of CHO-K1 cells with lipid mixing measured by fluorescence assays, using liposomes or complexes with plasmid DNA of the cationic lipids 1,2 dioleolyl-3-N, N, N,-trimethylammonium-propane (DOTAP), N-[2,3-(dioleoyloxy)propyl]-N, N, N,-trimethylammonium (DOTMA), or combinations of these lipids with dioleoylphosphatidylethanolamine (DOPE), at various lipid/DNA charge ratios. Mixing of the lipids of the complexes or liposomes with cellular membranes occurred readily at 37 degrees C, and was more efficient with liposomes than with complexes. Lipid mixing was inhibited at low temperatures (0-17 degrees C), by the presence of NH(4)Cl in the medium, and by low extracellular pH, indicating the involvement of the endocytic pathway in entry. In the absence of DOPE, there was no correlation between the efficiency of lipid mixing and the efficiency of transfection. Moreover, although DOPE, which is thought to promote membrane fusion, enhanced transfection, it did not always enhance lipid mixing. Neither the size nor the zeta potential of the complexes were clearly associated with transfection efficiency. Therefore, although fusion between the lipids of the complexes and cellular membranes takes place, a step at a later stage in the transfection process determines the efficiency of transfection.

Ammonium Chloride↗
PubMed1999

Stabilized plasmid-lipid particles: factors influencing plasmid entrapment and transfection properties.

Previous work has shown that plasmid DNA can be encapsulated in small 'stabilized plasmid-lipid particles' (SPLP) composed of 1, 2-dioleyl-3-phosphatidylethanolamine (DOPE), the cationic lipid N, N-dioleyl-N,N-dimethylammonium chloride (DODAC) and poly(ethylene glycol) (PEG) conjugated ceramides (PEG-Cer), employing a detergent dialysis procedure. These SPLP have potential as vectors for in vivo gene therapy. This study is aimed at characterizing the influence of the cationic lipid and PEG-Cer species on SPLP formation and in vitro transfection properties. It is shown that the transfection potency of SPLP is sensitive to the cationic lipid species employed, the size of the PEG polymer incorporated in the PEG-ceramide and the length of the acyl chain contained in the ceramide anchor. With regard to the influence of cationic lipid, the transfection levels achieved were highest for SPLP containing N-[2, 3-(dioleyloxy)propyl]-N,N-dimethyl-N-cyanomethylammonium chloride (DODMA-AN) and lowest for SPLP containing 3-beta-[N-(N', N'-dimethylaminoethyl)carbamoyl]-cholesterol (DC-CHOL), according to the series DODMA-AN>N-[2,3-(dioleyloxy)propyl]-N,N, N-trimethylammonium chloride (DOTMA)>DODAC>N,N-distearyl-N, N-dimethylammonium chloride (DSDAC)>DC-CHOL. Incorporation of short (PEG(750)) PEG polymers in the PEG-ceramide components resulted in modest improvements in transfection levels over PEG(2000) and PEG(5000) polymers, however variation of the length of the acyl chain contained in the hydrophobic ceramide anchor from octanoyl (PEG-CerC(8)) to myristoyl (PEG-CerC(14)) to arachidoyl (PEG-CerC(20)) had the most dramatic effects. Transfection levels achieved for SPLP containing PEG-CerC(8) were substantially larger than observed for SPLP containing PEG-CerC(14) or PEG-CerC(20), consistent with a requirement for the PEG-ceramide to dissociate from the SPLP surface for maximum transfection potency. It is also shown that the ability of SPLP to be accumulated into cells is a dominant factor influencing transfection potency, and that the transfection potency of SPLP that are accumulated is at least equivalent to that of cationic lipid-plasmid DNA complexes.

Cell Line↗
PubMed1998

Determination of deoxyribonucleoside triphosphate pool sizes in ribonucleotide reductase cDNA transfected human KB cells.

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis, which is responsible for controlling deoxyribonucleoside triphosphate (dNTP) pool size. It has been shown that transfection of RR M2 cDNA in human KB cells (M2-D clone) results in overexpression for the M2 subunit and resistance to hydroxyurea (HU). In this study, dNTP pool assays were performed to measure the pool sizes in six cell lines: two controls, three transfectants, and drug-induced HU-resistant (HUR) cells. Total dNTP levels among the six cell lines rose in the following order: KB wild-type, KB vector-only transfectant, M1 cDNA transfectant, M2 cDNA transfectant, M1/M2 cDNA transfectant, and HU-induced resistant clone. The dCTP levels of the cells mimicked the total dNTP pools on a smaller scale. The significant increases in the dCTP pool sizes of the M2-D, X-D, and HUR clones were proportional to their respective increases in RR activity. Relative to all other transfectants, the M1-D clone demonstrated lower dCTP levels but increased dATP pools. The M1-D clone demonstrated a significant resistance to dNTP inhibition of RR activity compared with the control KB wild-type cells. In contrast, a profound inhibition of dCTP and a decreased sensitivity to dATP inhibition was observed in M2-D, X-D, and HUR clones. In summary, M2 cDNA transfectants and HUR clones had increased RR activity as well as expanded dNTP pools, particularly dCTP, when compared with wild-type KB cells. These data provide evidence for the intertwined relationship between RR activity and dNTP pools.

Clone Cells↗
PubMed1998

Chemoprotective functions of glutathione S-transferases in cell lines induced to express specific isozymes by stable transfection.

The authors have shown that expression of mGSTM1-1 or hGSTP1-1 in MCF-7 cells protects against DNA alkylation by 4-nitroquinoline-1-oxide (NQO) in an isozyme-specific manner and is commensurate with relative specific activity. Expression of GSTs also conferred protection against both DNA strand breaks and sister-chromatid exchange induced by NQO. Interestingly, GST expression did not protect against NQO cytotoxicity in transfected MCF-7 cell lines, although resistance to NQO cytotoxicity was observed in a T47D pi transfectant line, expressing much higher specific activity of the transfected hGSTP1-1. However, high level expression of hGSTP1-1 or mGSTM1-1 in V79 transfectants did not confer resistance to cytotoxicity, indicating that expression of GST alone is not sufficient. The authors have also shown protection against AFB1 in cell lines expressing transfected rat CYP2B1 (V79MZr2B1) and transfected mGST-Yc (mGSTA3-3). Protection was observed against both alkylation of DNA (3-fold) by [3H]AFB1 and against AFB1 cytotoxicity (7-fold). Similarly, V79MZr1A1 cells that express CYP1A1 and either transfected human or murine GSTP1-1 (< 5000 mIU/mg, CDNB) exhibited > 70% decrease in covalent labeling of total nucleic acids by [3H]BPDE. However, no protection against the cytotoxicity of BPDE was conferred by expression of hGSTP1-1. Overall, these results indicate that in some (NQO or BPDE), but not all (AFB1) cases, protection by GST expression against DNA damage is more effective than protection against cytotoxicity. In addition, there is evidence to indicate that additional factor(s) other than high GST isozyme expression level and good substrate efficacy affect the degree of protection against cytotoxicity of reactive electrophiles. This includes the differential protection against NQO cytotoxicity in T47D pi, but not V79 Xh pi-33 cells and also the recent studies which showed that expression of the MRP GS-X conjugate efflux transporter confers synergistic protection against NQO cytotoxicity when co-expressed with transfected human GSTP1-1 in MCF-7 cells. Thus, protective efficacy conferred by GST expression can vary with different cellular targets and/or experimental end-points, as well as with variations in relative specific activity or in different cellular phenotypic contexts.

4-Nitroquinoline-1-oxide↗
PubMed1997

Novel cationic liposomes for DNA-transfection with high efficiency and low toxicity.

Liposomes containing the natural cationic amphiphile, sphingosine and some of its derivatives were used for transfection of DNA in vitro. Multilamellar liposomes comprised of dioleoylphosphatidylethanolamine (DOPE), different sphingosine derivatives, and diacylglycerols with varying fatty acid chains, preincubated with DNA, transfected efficiently the KK-1 murine granulosa cells. Most efficient transfection on this cell line was achieved with liposomes composed of phytosphingosine, DOPE, and dioctanoylglycerol (DC8G) (64:31:4.8, molar stoichiometry), which gave expression of the transfected gene 2-10-fold higher than the commercial reagent Lipofectin. At higher doses the new liposomes also caused markedly less cell death of KK-1 cells. On COS-7 cells these liposomes showed slightly, but significantly lower transfection, of approximately 70%, of that gained with Lipofectin. The murine Sertoli cells, MSC-1, selectively resisted transfection by the sphingosine derivative based liposomes tested, giving only 11-14% of the expression detected in Lipofectin transfected cells of the same line. In conclusion, the novel liposomes formulated offer an effective, technically easy and economical method of transfection for a variety of cultured cell lines.

Animals↗