Effects of pulse length and pulse strength on transfection by electroporation

The relative importance of pulse field strength E and pulse length tau 1/2 (half decay time of an exponential decay pulse) on the stable transfection frequency for HeLa or HUT-78 cells was investigated. Cells were transfected with plasmids containing the promoter and drug resistant genes pRSVgpt or pRSVneo by electroporation. The stable transfection frequency was assayed using the marker rescue technique. The transfection frequency increases with increasing values of E tau 1/2. For a given pulse length, the transfection frequency is proportional to the power of the pulse (E2 tau 1/2). Pulses with half decay times of 2.2 to 4.6 ms appear to be more efficient than 0.275 to 0.31 ms for stable transfection of HeLa cells.     

Enhancement of DNA-mediated gene transfer by inhibitors of autophagic-lysosomal function

A variety of compounds, known to influence the intravesicular transport and degradation of macromolecules, was studied for their effect on the efficiency of DNA-mediated gene transfer (transfection). The efficiency of transfection was measured by transformation of rat 2 thymidine kinase-deficient (tk^?) cells by the cloned herpes simplex I thymidine kinase gene (pAGO). When salmon sperm DNA (average molecular weight, 6 × 10⁶ D) was used as a carrier, the presence of either 20 mM NH₄Cl, 1 μM carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), or 5 mM 3-methyl adenine (3-MA) in the medium during incubation of the cells with the DNA-calcium-phosphate (DNA-Ca-P_i) precipitate, enhanced the efficiency of transfection by a factor of 10. If rat thymus DNA (greater than 30 × 10⁶ D) was used as a carrier, the transformation efficiency was much higher than with salmon sperm DNA. However, in this case treatment with 3-MA, NH₄Cl and FCCP enhanced the transformation frequency by slightly less than a factor of two. 3-MA further increased the transfection frequency if the cells were incubated with the compound after removal of the DNA-Ca-P_i coprecipitate, whereas NH₄Cl and FCCP had no such effect. Our results strongly suggest that these inhibitors of intracellular degradation can increase the frequency of transformation by increasing the cytoplasmic levels of exogenous DNA.     

Protoplast-transfection of Streptomyces chartreusis SF1623 with Actinophage Φr5 DNA

To establish a procedure for high frequency transfection in streptomycetes, the conditions and factors affecting the polyethyleneglycol (PEG) mediated transfection of S. chartreusis SF1623 by actinophage Φr5 DNA were studied. Protoplasts of S. chartreusis SF1623 prepared by treatment with lysozyme and achromopeptidase were very stable. Protoplasts from 20 to 22hr culture cells were more competent for transfection. The optimal pH of the medium for transfection was pH 7.6. The presence of NaCl, thymidine, ATP, ADP or adenosine in the transfection medium enhanced the frequency of transfection. The optimal conditions determined for protoplast transfection were 12.5% PEG 4,000, 300 mm NaCl, 1 mm thymidine, final concentration, Φr5 DNA and protoplasts in P3 medium (pH 7.6). The frequency of transfection under the optimal conditions was 5 × 10⁵ per μg Φr5 DNA and was about 3 × 10^?3 per regenerated protoplasts.

Progenitively mature phages appeared 4hr after incubation in the regeneration solution and their number continued to increase for about 11 hr. The burst size was estimated to be about 400.     

Study of mechanisms of electric field-induced DNA transfection. II. Transfection by low-amplitude, low-frequency alternating electric fields.

Electroporation for DNA transfection generally uses short intense electric pulses (direct current of kilovolts per centimeter, microseconds to milliseconds), or intense dc shifted radio-frequency oscillating fields. These methods, while remarkably effective, often cause death of certain cell populations. Previously it was shown that a completely reversible, high ionic permeation state of membranes could be induced by a low-frequency alternating electric field (ac) with a strength one-tenth, or less, of the critical breakdown voltage of the cell membrane (Teissie, J., and T. Y. Tsong. 1981. J. Physiol. (Paris). 77:1043-1053). We report the transfection of E. coli (JM105) by plasmid PUC18 DNA, which carries an ampicillin-resistance gene, using low-amplitude, low-frequency ac fields. E. coli transformants confer the ampicillin resistance and the efficiency of the transfection can be conveniently assayed by counting colonies in a selection medium containing ampicillin. For the range of ac fields employed (peak-to-peak amplitude 50-200 V/cm, frequency 0.1 Hz-1 MHz, duration 1-100 s), 100% of the E. coli survived the electric field treatment. Transfection efficiencies varied with field strength and frequency, and as high as 1 x 10(5)/micrograms DNA was obtained with a 200 V/cm square wave, 1 Hz ac field, 30 s exposure time, when the DNA/cell ratio was 50-75. Control samples gave a background transfection of much less than 10/micrograms DNA. With a square wave ac field, the transfection efficiency showed a frequency window: the optimal frequency was 1 Hz with a 200 V/cm field, and was approximately 0.1 Hz with a 50 V/cm field.(ABSTRACT TRUNCATED AT 250 WORDS)     

AP-4F,antennapedia peptide linked to an amphipathic alpha helical peptide,increases the efficiency of Lipofectamine-mediated gene transfection in endothelial cells

Typically, endothelial cells are difficult to transfect. In this study, we report that antennapedia peptide (AP) linked to L-4F, a water-soluble, amphipathic alpha helical peptide that avidly binds lipids (AP-4F) increases Lipofectamine 2000-mediated transfection of bovine coronary endothelial cell cultures. Transfection efficiency was monitored by flow cytometry and fluorescent microscopy. Lipofectamine 2000 transfection of endothelial cell cultures with green fluorescence protein (GFP)-DNA typically yields transfection efficiencies of 35.4+/-3.3% with low levels of cell death (8.1+/-1.0%). Pre-treatment of the Lipofectamine 2000-GFP-DNA complexes with AP-4F for 5 min increased transfection to 58.2+/-2.8% without increasing cell death. AP-4F increases Lipofectamine 2000-mediated transfection in a time-dependent fashion (within 10-20 min). Systematic studies reveal that the individual components of AP-4F, i.e., AP and L-4F alone, are ineffective in increasing Lipofectamine 2000-mediated transfection and that AP-4F must be directly associated with DNA liposomes prior to transfection for optimal uptake by endothelial cells. These observations demonstrate that AP-4F may be useful for increasing the transfection efficiency of endothelial cell cultures with standard commercially available reagents.     

High-frequency Protoplast-transfection of Streptomyces parvulus 2297 with Actinophage R4 DNA

High frequency transfection of Streptomyces parvulus with actinophage R4 DNA was performed by modifying the procedure of protoplast transformation of S. coelicolor A3(2) with SCP2 plasmid DNA [Bibb et al., Nature, 274, 398 (1978)]. Optimum conditions for protoplast transfection included the presence of 16~24% (w/v) polyethyleneglycol 4000, and the maximum efficiency of transfection was 3 × 10^?5 per phage DNA molecule. This value was at least 100 times higher than the efficiency of previously reported transfection systems in Streptomyces.     

Mechanical strain increases gene transfer to skeletal muscle cells

Gene transfer techniques possess tremendous potential for treating diseases and for facilitating the study of basic physiological processes. However, further development of efficient and safe methods for gene transfer is needed. The purpose of this study was to test the hypothesis that mechanical strain increases the transfer of DNA to differentiated skeletal muscle cells. We tested this hypothesis by applying cyclic strain to cultured skeletal myotubes either prior to or immediately after the introduction of exogenous DNA complexed with lipids, with strains of varying magnitude (10%, 20% and 30%), number (1800, 3600 and 7200 strain cycles) and frequency (0.5, 1.0 and 1.5 Hz). Results demonstrated that DNA transfection was increased by exposing muscle cells to cyclic strain, and that strain magnitude, number and frequency each influenced DNA transfection. Optimal strain conditions (20% strain magnitude, 3600 cycles applied at 1 Hz) were utilized to examine the role of membrane transport systems in strain-induced increases in DNA transfection. Filipin III was used to inhibit caveolar transport and was found to inhibit strain-mediated increases in DNA transfection, whereas chlorpromazine, used to inhibit clathrin-coated vesicle transport, had no effect. These results indicate that mechanical strain may be an effective method for increasing DNA transfection in skeletal muscle through enhanced caveolar transport.     

SV 40-transformed normal and DNA-repair-deficient human fibroblasts can be transfected with high frequency but retain only limited amounts of integrated DNA

The ability of simian virus 40-transformed human fibroblasts to integrate and maintain transfected genomic DNA has been investigated in two normal and six DNA-repair-deficient human cell lines. These cell lines were transfected with DNA containing two selective markers (G418 and hygromycin (Hyg) resistance) separated by random pieces of human DNA of 0-40 kb in length. The transfection frequency for the selected (G418R) marker was between 2 x 10(-4) and 2 x 10(-3) for all cell lines, comparable to many other mammalian systems. About 50% of the G418R colonies were also initially resistant to Hyg. Analysis of the DNA from individual clones expanded for a further month revealed, however, that about one to three copies of the selected marker but only about 0.1 copy per cell of the unselected marker were maintained. Our results were broadly similar for all eight cell lines. Thus the amount of integrated DNA that is stably maintained in these cells is in general very small (less than 50 kb). This may provide an explanation for the difficulties encountered in many laboratories in attempts to correct the defect in DNA-repair-deficient human cells by transfection with genomic DNA. Our results also show that none of several defects in DNA repair has any obvious effect on either the transfection frequency or the amount of stably integrated foreign DNA.     

鸡原始生殖细胞转染条件优化

为获得鸡原始生殖细胞(primordial germ cells,PGCs)的最佳转染效率,本研究比较不同质粒用量和不同细胞数在3种转染试剂(Lipofectamine 2000、3000和LTX&Plus Reagent)中PGCs的转染效率,利用荧光激活细胞分选技术(fluorescence activated cell sorting technology,FACS)辅助优化Lipofectamine 3000转染试剂,经FACS进一步分选获得带绿色荧光蛋白(GFP)的PGCs,继续培养3周后,移植回注到受体鸡胚中,移植3.5 d后分离性腺拍照观察。结果显示,转染试剂Lipofectamine 3000的转染效率最高,质粒、Lipofectamine 3000转染试剂和PGCs细胞数的配比为3μg:4μL:0.5×10⁴个,转染5h转染效率最高,达到23.4%,与现有的研究结果相比提高了2倍以上。移植回注PGCs到受体鸡胚中,荧光显微镜观察到鸡胚性腺中有GFP阳性细胞。本研究综合考虑转染试剂、质粒用量和细胞数量的影响因素以优化PGCs的转染条件,为高...     

Targeting of lipid-protamine-DNA (LPD) lipopolyplexes using RGD motifs

The incorporation of pegylated lipid into Lipid-Protamine-DNA (LPD-PEG) lipopolyplexes causes a decrease of their in vitro transfection activity. This can be partially attributed to a reduction in particle binding to cells. To restore particle binding and specifically target LPD formulations to tumor cells, the lipid-peptide conjugate DSPE-PEG5K-succinyl-ACDCRGDCFCG-COOH (DSPE-PEG5K-RGD-4C) was generated and incorporated into LPD formulations (LPD-PEG-RGD). LPD-PEG-RGD was characterized with respect to its biophysical and biological properties. The Incorporation of DSPE-PEG5K-RGD-4C ligands into LPD formulations results in a 5 and a 15 fold increase in the LPD-PEG-RGD binding and uptake, respectively, over an LPD-PEG formulation. Enhancement of binding and uptake resulted in a 100 fold enhancement of transfection activity. Moreover, this transfection enhancement was specific to cells expressing appropriate integrin receptors (MDA-MB-231). Huh7 cells, known for their low level of alphavbeta3 and alphavbeta5 integrin expression, failed to show RGD mediated transfection enhancement. This transfection enhancement can be abolished in a competitive manner using free RGD peptide, but not an RGE control peptide. Results demonstrated RGD mediated enhanced LPD-PEG cell binding and transfection in cells expressing the integrin receptor. These formulations provide the basis for effective, targeted, systemic gene delivery.     

Polyethylene glycol-dependent transfection of Acholeplasma laidlawii with mycoplasma virus L2 DNA

Phenol-extracted DNA from mycoplasma virus L2 was able to transfect Acholeplasma laidlawii in the presence of polyethylene glycol. Transfection was sensitive to DNase and was most efficient with 36% (wt/vol) polyethylene glycol 8000 and cells in logarithmic growth. Virus production by the transfected cells was similar to that of the cells infected by intact virus. L2 DNA transfected A. laidlawii with a single-hit dose-response curve, reaching saturation at high DNA concentrations. Optimum transfection frequencies were about 10(-7) transfectants per L2 DNA molecule and 10(-4) transfectants per CFU. When DNA was present in saturating amounts, the number of transfectants increased linearly with the number of CFU present in the transfection mixture, suggesting that DNA uptake does not occur by a mechanism involving cell fusion. The cleavage of the superhelical mycoplasma virus L2 genome with restriction endonucleases that cleave the DNA molecule once reduced the transfection frequency. Host cell modification and restriction of transfecting L2 DNA were similar to those for infecting L2 virions.     

Muscle transfection and permeabilization induced by electrotransfer or pluronic® L64: Paired study by optical imaging and MRI

Background

Muscle transfection by electrotranfer is an efficient currently used procedure. Recently, the block copolymer pluronic L64 has been reported to improve muscle transfection. Both procedures are known to permeabilize muscle fibres. Relation between muscle transfection and permeabilization by electrotransfer and L64 was investigated herein.

Methods

Muscle transfection was evaluated by optical detection of the luciferase reporter gene activity. Muscle permeabilization was evaluated by the uptake of the T1 contrast agent gadolinium-Dotarem (Gd-DOTA) using Magnetic resonance imaging (MRI). Histological examination of muscle sections was also performed.

Results

Electrotransfer and L64 (at a 0.25% concentration) similarly improved muscle transfection, although the interindividual variability was higher for pluronic. On the same animals, the permeabilized volume to the Gd-DOTA was significantly increased after electrotransfer, and L64 from 0.1% to 1%. The concentration of the Gd-DOTA in the permeabilized volume was significantly increased after electrotransfer and L64 at 0.5% and 1%. By histological observation, the inflammation was maximum at day 3 after electrotransfer or L64 injection, and mostly reversed after 7 days. The permeabilized volume and the transfection level correlated for the set of all the conditions tested. However, no significant correlation was observed between Gd-DOTA concentration and transfection.

General significance

It is possible to use successively on the same animals MRI and optical imaging for paired studies of muscle transfection and permeabilization. Permeabilization is possibly not related to gene transfer but it indicates membrane modification related to transfection by the electrotransfer or co-injection of DNA with the L64.     

Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.     

Verapamil enhances the efficiency of DNA-mediated gene transfer in mammalian cells

Treatment of Ltk^? cells with the calcium antagonists, verapamil and diltiazem, but not nifedipin, causes a 3-fold enhancement of the frequency of transfer of the cloned gene for herpes simplex virus thymidine kinase (HSV-tk). The frequency of phenotypic expression of the HSV-tk DNA was 20 to 34 times higher than that of genotypic transformation. Phenotypic expression was also 2.3 to 2.6 times increased when 20 μg/ml of verapamil was present during calcium phosphate-mediated DNA transfection.     

Enhancement of gene delivery by an analogue of alpha-MSH in a receptor-independent fashion

In order to transfect melanoma specifically by receptor-mediated endocytosis we prepared dioctadecyl aminoglycylspermine (lipospermine)--DNA complexes with [Nle(4),D-Phe(7)]-alpha-MSH(4--10), a pseudo-peptide analogue of alpha-melanocyte stimulating hormone (alpha-MSH) linked to a thiol-reactive phospholipid. With these complexes we obtained an up to 70-fold increase of transfection with B16-F1 melanoma cells. However when B16-G4F, an alpha-MSH receptor negative melanoma cell line was transfected, an up to 700-fold increased transfection efficiency was observed. The peptide hormone analogue was equally efficient when it was only mixed with lipospermine--DNA complexes without covalent coupling. In addition to melanoma cells we also obtained up to 30-fold increased transfection with BN cells (embryonic liver cells). Our data show that an alpha-MSH analogue increased transfection independently of the MSH receptor expression but reaches efficiencies approaching those obtained with peptides derived from viral fusion proteins. The absence of targeting of constructs containing [Nle(4),D-Phe(7)]-alpha-MSH(4-10) can probably be attributed due to the relatively modest number of MSH receptors at the surface of melanoma. We suggest, however, that the peptide hormone analogue used in this study has membrane-active properties and could be of interest as helper agent to enhance non-viral gene delivery presumably by endosomal-destabilizing properties.     

Comparison of spontaneous and ultraviolet-induced mutagenesis on naked SV40 DNA and SV40 virus

Molecular aspects of mutagenesis in mammalian cells have been essentially analyzed using biological probes such as viruses and shuttle vector. Although the main data concerning the specificity of carcinogen-induced mutations are similar, the observed spontaneous mutation frequencies are significantly different when using one or the other model. This frequency is considerably higher with shuttle vectors than with viruses. We have performed an analysis of mutagenesis in order to determine if the obligatory transfection step associated with shuttle vector technology was responsible for the high mutation frequency found with these molecules. For this purpose simian virus 40 (SV40) genome used as virus or as naked DNA was introduced into permissive cells by viral infection or DNA transfection respectively. Our results show that transfection alone does not induce a higher mutation frequency on SV40 DNA the virus infection. Moreover, we have shown that the ultraviolet-light induced mutation spectrum was similar on the SV40 VP1 gene after viral infection or DNA transfection.     

Detection by flow cytometry of protoplast fusion and transient expression of transferred heterologous CD4 sequences in COS-7 cells

Transfer and expression of a plasmid containing the gene encoding the human T-cell antigen CD4 by protoplast fusion was measured by flow cytometry (FCM). Protoplasts were prelabeled with fluorescein isothiocyanate (FITC) and fused to COS-7 cells. Nonspecific protoplast adsorption to the plasma membrane was differentiated from successful protoplast fusion by the addition of an antibody directed against fluorescein to quench extracellular protoplast fluorescence. Transfection efficiencies were defined as both percent CD4 expressing cells and CD4 expression levels on a single cell basis in the transient immunofluorescence assay. Cell sorting studies indicated that intracellular protoplast-associated fluorescence immediately after fusion exhibited a good correlation with transient CD4 transfection efficiencies as measured by indirect immunofluorescence. Reconstruction experiments comparing CD4 transfer efficiencies of protoplast fusion and calcium phosphate transfection showed that fusion resulted in a higher percentage of CD4 expressing transfectants, while calcium phosphate transfection yielded higher CD4 expression levels on a single cell basis. Thus, FCM appears to be useful as a new tool for sensitive detection of transient expression of heterologous reporter genes in COS-7 cells.     

Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells.

We describe the construction of an adeno-associated virus (AAV) vector in which the coding sequence of the procaryotic gene neo is expressed under the control of the major AAV promoter p40. This AAV-neo vector allowed stable expression of neo as a dominant selective marker in mammalian cells by selection of cells which were resistant to the antibiotic geneticin (G418). When the vector was introduced into human (293 or HeLa) cell lines by a DNA transfection procedure, stable geneticin-resistant colonies were obtained. When the vector was first packaged into AAV particles and then introduced into cells via particle infection, geneticin-resistant cells were obtained at higher frequencies than those obtained by DNA transfection. In geneticin-resistant cells the AAV-neo vector was integrated at low copy number and could be rescued by subsequent infection with wild-type AAV and the helper adenovirus or, in some cases, by infection with adenovirus alone. The rescued AAV-neo vector could then be recovered as amplified unintegrated DNA from a Hirt lysate. These results demonstrate that AAV can be used as a transducing viral vector for stable integration and expression of a foreign gene in mammalian cells. The high frequency of integration and the ability to rescue the integrated vector suggest that this vector system may be useful for selecting genes from cDNA libraries. This vector may also be useful for introduction of genes into cells which are refractory to transfection in procedures such as those involving the use of CaPO4 or DEAE-dextran.     

Error-prone mutagenesis detected in mammalian cells by a shuttle vector containing the supF gene of Escherichia coli.

When a shuttle vector containing a tyrosine suppressor tRNA (supF) gene as a target for mutagenesis replicated in a monkey kidney cell line, the frequency of SupF+ mutations was 2.3 +/- 0.5 x 10(-3). When the host cells were treated with ethyl methanesulfonate 40 h before transfection, a 10-fold increase in SupF+ mutation frequency was observed. These results supported the hypothesis that a damage-inducible mutagenic pathway exists in mammalian cells and also demonstrated the utility of this shuttle vector for the study of mutagenesis in mammalian cells.