A novel method of transfection of marine bacteria Pseudoalteromonas espejiana with deoxyribonucleic acid of bacteriophage PM2

DNA of bacteriophage PM2 is a convenient test object for studying DNA-damaging genotoxic agents. The extent of DNA damage can be estimated by the ability of damaged DNA for transfection of host cells, marine bacterium Pseudoalteromonas espejiana (Pae), str. BAL-31. The efficiency of transfection of Pae lines maintained for long periods without freezing was found to be very low upon the use of a widely accepted transfection method developed by van der Schans et al. (1971). Such cultures grown in a medium with 10 mM Ca²⁺ standard for Pae contained cell aggregates and exopolymer material. Pae was found to be capable of growing in a medium without the calcium supplement in the presence of chelator EGTA (low-calcium medium, LCM). After growth in LCM, cells did not aggregate, cultures lacked the activity of nuclease BAL, and transfection efficiency of cells grown in LCM drastically increased. Based on these results, a novel procedure of transfection with an efficiency of 2 × 10⁴?2 × 10⁵ infectious centers per microgram of PM2 DNA was developed.     

Growth and differentiation of stage 24 limb mesenchyme cells in a serum-free chemically defined medium

A serum-free defined medium which supports the differentiation of chick limb mesenchymal cells has been developed. In this medium, stage 24 embryonic limb mesenchymal cells which are plated at high density (5 x 10(6) cells/35-mm culture dish) differentiate into chondrocytes. Morphologically, these cultures appear only slightly different from those in which the cells are maintained in serum-containing medium. DNA levels and proline incorporation in cultures grown in defined medium are indistinguishable from control cultures. The rate of radiolabeled sulfate incorporation, a monitor of the rate of proteoglycan synthesis, in Day 8 high-density cultures maintained in defined medium is approximately 70-80% of control values. Additionally, growth and differentiation of intermediate-density (2 x 10(6) cells/35-mm culture dish) and low-density (1 x 10(6) cells/35-mm dish) cultures are also supported by this defined medium. The availability of this medium allows exploration of bioactive factors which affect or modulate mesenchymal cell differentiation and subsequent development.     

Resistance to DNA denaturation in irradiated Chinese hamster V79 fibroblasts is linked to cell shape

Exponentially growing Chinese hamster V79-171b lung fibroblasts seeded at high density on plastic (approximately 7 x 10(3) cells/cm2) flatten, elongate, and produce significant amounts of extracellular fibronectin. When lysed in weak alkali/high salt, the rate of DNA denaturation following exposure to ionizing radiation is exponential. Conversely, cells plated at low density (approximately 7 x 10(2) cells/cm2) on plastic are more rounded 24 h later, produce little extracellular fibronectin, and display unusual DNA denaturation kinetics after X-irradiation. DNA in these cells resists denaturation, as though "constraints" to DNA unwinding have developed. Cell doubling time and distribution of cells in the growth cycle are identical for both high and low density cultures as is cell survival in response to radiation damage. The connection between DNA conformation and cell shape was examined further in low density cultures grown in conditioned medium. Under these conditions, cells at low density were able to elongate, and DNA denaturation of low density cultures was identical to that of high density cultures. Conversely, cytochalasin D, which interferes with actin polymerization causing cells to "round up" and release fibronectin, allowed development of constraints in high density cultures. These results suggest that DNA conformation is sensitive to changes in cell shape which result when cells are grown in different environments. However, these changes in DNA conformation detected by the DNA unwinding assay do not appear to play a direct role in radiation-induced cell killing.     

Study of mechanisms of electric field-induced DNA transfection. I. DNA entry by surface binding and diffusion through membrane pores.

A study of mechanisms of electrotransfection using Escherichia coli (JM 105) and the plasmid DNA pBR322 as model system is reported. pBR322 DNA carries an ampicillin resistance gene: E. coli transformants are conveniently assayed by counting colonies in a selection medium containing 50 micrograms/ml ampicillin and 25 micrograms/ml streptomycin. Samples not exposed to the electric field showed no transfection. In the absence of added cations, the plasmid DNA remains in solution and the efficiency of the transfection was 2 x 10(6)/micrograms DNA for cells treated with a 8-kV/cm, 1-ms electric pulse (square wave). DNA binding to the cell membrane greatly enhanced the efficiency of the transfection and this binding was increased by milimolar concentrations of CaCl2, MgCl2, or NaCl (CaCl2 greater than MgCl2 greater than NaCl). For example, in the presence of 2.5 mM CaCl2, 55% of the DNA added bound to E. coli and the transfection efficiency was elevated by two orders of magnitude (2 x 10(8)/micrograms DNA). These ions did not cause cell aggregation. With a low ratio of DNA to cells (less than 1 copy/cell), transfection efficiency correlated with the amount of DNA bound to the cell surface irrespective of salts. When the DNA binding ratio approached zero, the transfection efficiency was reduced by two to three orders, indicating that DNA entry by diffusion through the bulk solution was less than 1%. Square pulses of up to 12 kV/cm and 1 ms were used in the electrotransfection experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum-free large-scale transient transfection of CHO cells

To date, methods for large-scale transient gene expression (TGE) in cultivated mammalian cells have focused on two transfection vehicles: polyethylenimine (PEI) and calcium phosphate (CaPi). Both have been shown to result in high transfection efficiencies at scales beyond 10 L. Unfortunately, both approaches yield higher levels of recombinant protein (r-protein) in the presence of serum than in its absence. Since serum is a major cost factor and an obstacle to protein purification, our goal was to develop a large-scale TGE process for Chinese hamster ovary (CHO) cells in the absence of serum. CHO-DG44 cells were cultivated and transfected in a chemically defined medium using linear 25 kDa PEI as a transfection vehicle. Parameters that were optimized included the DNA amount, the DNA-to-PEI ratio, the timing and solution conditions for complex formation, the transfection medium, and the cell density at the time of transfection. The highest levels of r-protein expression were observed when cultures at a density of 2.0 x 10(6) cells/ml were transfected with 2.5 microg/ml DNA in RPMI 1640 medium containing 25 mM HEPES at pH 7.1. The transfection complex was formed at a DNA:PEI ratio of 1:2 (w/w) in 150 mM NaCl with a 10-min incubation at room temperature prior to addition to the culture. The procedure was scaled up for a 20-L bioreactor, yielding expression levels of 10     

Interferon induction in marrow-derived macrophages: regulation by L cell conditioned medium

Mouse bone marrow cells grown in medium enriched with L cell conditioned medium (LCM) as a source of colony stimulating factor (CSF) yield populations of adherent macrophages which are quite sensitive to induction of interferon (IFN) by viral and nonviral inducers. We examined the role of LCM in the sensitivity of marrow macrophage cultures to IFN induction. Removal of LCM from the cultures for as little as 3 hours markedly reduced the IFN titers induced by a double stranded ribopolynucleotide (poly I:C) or a lipopolysaccharide (LPS), while induction by Newcastle disease virus (NDV) was unaffected. Addition of anti-CSF serum to LCM medium also reduced IFN titers in response to polyI:C but had no effect on NDV induction. The inhibitory effect of anit-CSF indicates that the LCM requirement is at least partially related to the colony stimulating activity of the medium. We postulate that CSF regulates the initial interaction of macrophages with polyI:C or LPS rather than the synthesis and secretion of interferon by the phagocytes. Nearly complete restoration of IFN induction with polyI:C was obtained when LCM deprived cultures were reincubated with LCM medium previously conditioned by marrow cultures.     

Transfection of Escherichia coli Spheroplasts III. Facilitation of Transfection and Stabilization of Spheroplasts by Different Basic Polymers

The only compound which fully replaced protamine sulfate in facilitating transfection of Escherichia coli spheroplasts by phage DNAs was spermine; poly-l-lysine, poly-l-arginine, DEAE-dextran, histones, and many other polyamines were only slightly effective. Higher-molecular-weight compounds were effective at lower concentrations, and each compound had a sharp concentration optimum. The specificity of the facilitation of transfection is discussed in light of Leonard and Cole's (1972) isolation of a polyamine- or protamine-like, natural competence factor from Streptococci. By standardizing growth conditions for spheroplast cultures, storing spheroplasts in minimal medium, and adding both protamine sulfate and polyamines to spheroplasts, reproducible competence levels were obtained. Thus, 95% of all spheroplast preparations gave efficiencies of transfection between 10(-3) and 3 x 10(-4) for lambda DNA; between 10(-6) and 3 x 10(-8) for T7 DNA; and between 3 x 10(-6) and 10(-7) for T5 phage DNA. The stability of the spheroplasts was extended from 10 h to between 2 and 5 days, depending on the DNA used for transfection.     

Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis.

Simian Cos-1 cells were transfected electrically with the plasmid pCH110 carrying the beta-galactosidase gene. The efficiency of transfection was determined by a transient expression of this gene. When the plasmid was introduced into a cell suspension 2 s after pulse application, the transfection efficiency was shown to be less than 1% as compared with a prepulse addition of DNA. Addition of DNAase to suspension immediately after a pulse did not decrease transfection efficiency, thus the time of DNA translocation was estimated to be less than 3 s. The use of electric treatment medium, in which the postpulse colloid-osmotic cell swelling was prevented, did not affect the transfection efficiency. These results contradict both assumptions of free DNA diffusion into cell through the long-lived pores and of involvement of osmotic effects in DNA translocation. Transfection of cells in monolayer on a porous film allowed creation of the spatial asymmetry of cell-plasmid interaction along the direction of electric field applied. A pulse with a polarity inducing DNA electrophoresis toward the cells resulted in the 10-fold excess of transfection efficiency compared with a pulse with reverse polarity. Ficoll (10%) which increases medium viscosity or Mg2+ ions (10 mM) which decrease the effective charge of DNA, both reduced transfection efficiency 2-3-fold. These results prove a significant role of DNA electrophoresis in the phenomenon considered. The permeability of cell membranes for an indifferent dye was shown to increase noticeably if the cells were pulsed in the presence of DNA. This indicates a possible interaction of DNA translocated with the pores in an electric field, that results in pore expansion.     

Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks

Methods were developed and evaluated for the preservation of tissue cells grown in suspension culture and the reestablishment of suspension cultures directly from inoculum stored at -175 C. The factors investigated were processing pH, temperature of processing, freezing medium, and method of inoculation of the starter suspension cultures from the frozen stock (-175 C). Three parameters, cell viability, cell size, and growth potential in suspension culture after freezing, were used to evaluate the various factors. The results indicate that cells processed at 4 C, frozen at 1 C per min to -50 C in a medium containing 5% dimethyl sulfoxide plus 10% bovine serum at concentrations of 2 x 10(7) to 4 x 10(7) cells/ml, and stored at -175 C will reestablish suspension cultures directly from frozen seed. A 1-ml amount of frozen stock inoculated into 99 ml of medium routinely produced 2 x 10(6) to 3 x 10(6) viable cells/ml (2 x 10(8) to 3 x 10(8) total cells) in suspension culture in 4 to 5 days. Inoculum preserved by this procedure grew equally well in either serum-free or serum-containing growth medium.     

Improvement of transformation and electroduction in avermectin high-producer,<Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

Factors affecting the PEG-mediated transformation and electrotransformation of Streptomyces avermitilis protoplasts, an industrial avermectin high-producer, were evaluated. The maximum protoplast transformation efficiency under optimum conditions with PEG was 3 x 106 transformants per microg plasmid pIJ702 DNA. The efficiency of electrotransformation with the same plasmid the intact cells grown in medium with 0.5 mmol/L CaCl2, suspended in buffer with 0.5 mol/L sucrose +1 mmol/L MgCl2, and pulsed at an electric field strength of 10 kV/cm, 800 ohms, 25 microF, was of 2 x 10(3) transformants per microg DNA. When the cells were electroporated after mild lysozyme-treatment, the efficiency was up to 10(4) transformants per microg DNA. Electroporation of protoplasts and germlings had a lower efficiency (10(2) transformants per microg DNA). We report that electroporation under optimum conditions can be used for direct transfer of nonconjugative plasmid pIJ699 between two different Streptomyces species, S. avermitilis and S. lividans.     

High-density transient gene expression in suspension-adapted 293 EBNA1 cells

Large-scale transient gene expression (TGE) in mammalian cells is an attractive method to rapidly produce recombinant proteins for pre-clinical studies, with some processes reported to reach 100 L. However, the yield remains low, hardly over 20 mg protein/L, mainly because the current TGEs have been performed at low cell density (approximately 5 x 10(5) cells/mL). In this study, the strategy to improve TGE focuses on facilitating transfection at high cell density. A high-density perfusion culture of 293 EBNA1 cells was established in 2-L bioreactor using Freestyle 293 expression medium (Invitrogen, Singapore) to grow the cells for transfection. Transfection was then carried out at 1 x 10(7) cells/mL using polyethylenimine (PEI) as DNA carrier, at the optimized conditions of 6 microg DNA/10(7) cells and 1:3 DNA to PEI mass ratio. During the post-transfection phase, 80.8 mg/L of the model protein, EPO was obtained at day 5.5 post-transfection (130 mg total EPO production) using a fed-batch culture mode. In comparison, perfusion cultures using an enriched SFM II medium resulted in a longer post-transfection production phase (8 days), and 227 mg of EPO was produced in 10.7 L medium, showing that high-density TGE enables the production of several hundreds of milligrams of protein in a 2 L bioreactor. In addition, a protocol for economical plasmid preparation based on anion exchange was also established to satisfy TGE's demand in terms of quality and quantity. To the best of our knowledge, this is the first report of transient transfections at a high cell density of up to 1 x 10(7) cells/mL.     

Frequency of infection and efficiency of transfection of Rhizobium meliloti cells and spheroplasts.

Competent cultures of Rhizobium meliloti cells and spheroplasts obtained by various methods were infected with DNA of phage 1A. The Frequency of infection among the cells and spheroplasts was 2 X 10(-8)-5 X 10(-10). The efficiency of transfection calculated from the ratio of plaque forming units to infective DNA molecule of phage 1A was 5 X 10(-8) to 10(-10). Frequency of infection and efficiency of transfection among the competent cells were by one order of magnitude higher in the case of the spheroplasts. The use of various media did not noticeably alter the efficiency of transfection.     

Properties of electroporation-mediated DNA transfer in Escherichia coli

Efficient and reproducible DNA-transfection was attained in E. coli, by electroporation. The yield of the transfectants was affected by pretreatment of the recipient cells as well as by the composition of the electroporation medium. Using a single pulse procedure, relationships among the electrical parameters, the transfection efficiency, and the cellular viability were investigated in 10 mM Tris-HCl buffer (pH 7.5) containing 5% sucrose. Certain sodium salts (e.g., citrate, phosphate, and sulfate) were promotive, whereas Mg2+, DEAE-dextran, and polyvinylpyrrolidone were inhibitory to the transfection. Heterologous nucleic acids (native DNA, denatured DNA, and tRNA) exerted only a marginal effect on transfection with a viral replicative-form DNA. The efficiency of DNA transfer was affected by culture conditions, and bacteria grown at a higher temperature were more competent. The electroporation system was more efficient than an improved CaCl2 method, not only in transfection with viral single- and double-stranded DNAs, but also in transformation with plasmid DNAs.     

Relationship Between Competence for Transfection and for Transformation

Deoxyribonucleic acid (DNA) extracted from phage SPP1 is highly infectious on Bacillus subtilis competent cells; the efficiency of infection is 5 x 10(3) to 6 x 10(3) phage equivalents per plaque-forming unit. This DNA was used to study the relationship between competence for transfection and for transformation. The experiments were concerned with the frequency of infection and transformation in mutants exhibiting different levels of competence, the effect of periodate on competence for infection and for transformation, the competition between phage and bacterial DNA, the transformation of cells preinfected with phage DNA, and the infection of cells pretreated with bacterial DNA. The data show that B. subtilis cells competent for transformation are also competent for transfection and vice versa; transfection with phage DNA represents, therefore, a simple way to measure the total number of competent cells in a culture. The fraction of competent cells, determined by SPP1 DNA infection, varied from 10(-2) to 7 x 10(-2).     

Conditions increasing the adrenergic properties of dissociated chick superior cervical ganglion neurons grown in long-term culture

Neurons dissociated from the embryonic chick superior cervical ganglion (SCG) were separated from ganglionic nonneuronal cells using a density gradient formed with Percoll. The sympathetic neurons were then grown for 3-4 weeks in serum containing medium on a polyornithine substrate precoated with heart-conditioned medium. Both catecholamine (CA) and acetylcholine (ACh) are synthesized and accumulated by these neurons, but the amount of CA is higher and increases much more over time in culture than the amount of ACh. The cultures become therefore more adrenergic with time. We report here that the adrenergic properties of these cells can be enhanced. A 3-fold increase in CA synthesis, as expressed on a per neuron basis, is obtained by increasing neuron cell density 3- to 4-fold. ACh synthesis, however, is decreased at high neuronal density. Optimal CA production is obtained at densities of 120-150,000 neurons/cm2. This effect is due to direct cell contact since it cannot be transferred to low density cultures by medium conditioned by high density cultures. Nerve growth factor concentrations 5-10-fold higher than the amount necessary for optimal neuronal survival (1 microgram/ml 7S NGF) increases CA production but do not affect ACh synthesis. This effect is highest at low plating densities (20-30,000 neurons/cm2, 10-fold increase) and progressively decreases with increasing neuronal density. No increase is obtained in high density cultures where CA production is maximal. In addition, we made the novel observation that medium conditioned by chick liver cells in culture (LCM) increases CA production approximately 4-fold, whereas it does not increase ACh production by the SCG neurons. Work is in progress to biochemically characterize the active component(s) present in the LCM and to determine whether they favor the survival of a subpopulation of adrenergic neurons possible present in these ganglia. Alternatively, the adrenergic differentiation of neurons initially capable of synthesizing both CA and ACh could be selectively increased by LCM.     

Studies on the development of metacyclic Trypanosoma brucei sspp. cultivated at 27 degrees C with insect cell lines

When transformed procyclic trypanosomes of three stocks of Trypanosoma brucei brucei and one stock of T.b. rhodesiense were grown at 27 degrees C in 25-cm2 flasks containing Anopheles gambiae cells, some of them developed into forms infective for mice. Infectivity titrations on trypanosome suspensions revealed that up to 2.8 X 10(5) metacyclic forms per ml could be produced, and the cultures remained infective for varying periods of up to 72 days when they were terminated. Of the various culture media tested, a mixture of three volumes of trypanosome medium and one volume of Anopheles medium was the most successful. Control cultures of trypanosomes grown in medium without cells were generally not infective, but two of the stocks gave rise to a few sporadic infections. Trypanosome populations could be subpassaged in the Anopheles cell cultures without loss of infectivity. Metacyclic forms separated from infective cultures by DEAE-cellulose columns had a surface coat.     

Isolation and characterization of a mildly thermophilic nonsulfur purple bacterium containing bacteriochlorophyll b

A method for transformation of whole Bacillus amyloliquefaciens cells by electroporation was developed. The procedure is as efficient as the protoplast transformation method, resulting in up to 10(5) transformants/micrograms plasmid DNA, but requires less effort and time. Cells for electroporation were grown to late exponential phase in a rich medium supplemented with 0.25 M sucrose, washed with and resuspended in 0.25 M sucrose, 1 mM HEPES, 1 mM MgCl2, 10% (v/v) glycerol, pH 7.0, at 3-5 x 10(10) cells/ml for storage at -80 degrees C. The highest transformation frequency was obtained at 7.5 kV/cm with a 25 microF capacitor. The transformation efficiency increased linearly with DNA concentration at least over the range 10 ng-12.5 micrograms/ml. Transformations with ligated DNA and of industrial strains were also successful. In addition, B. subtilis cells treated as above could be transformed by electroporation, resulting in 10(4) transformants/micrograms DNA at 12.5 kV/cm.     

Transfection of Lactobacillus bulgaricus protoplasts by bacteriophage DNA

A protoplast transfection system has been developed for Lactobacillus bulgaricus. The procedure involves a polyethylene glycol-mediated fusion of bacteriophage DNA encapsulated in liposomes into mutanolysin-treated cells. With L. bulgaricus B004 and DNA isolated from the phage phi c5004, transfection reached a maximum when at least 95% of the cells were osmotically fragile. The incorporation of phage DNA into liposomes was essential; no transfectants were detected in the absence of liposomes. The largest number of transfectants was observed after longer periods (20 min) of fusion of mutanolysin-treated cells and liposomes with polyethylene glycol. The maximum efficiency of 5 x 10(7) PFU/microgram of DNA was reached after a 24-h incubation in growth media prior to plating transfected cells in an agar overlay to detect the appearance of plaques. A minimum of 4 h of incubation in growth medium after fusion was required to detect the production and release of virions. The possibility that the high frequencies observed were due to bursting of transfected cells and subsequent infection of additional cells was found not to be a factor. The number of transfectants observed was directly proportional to the quantity of DNA added. These results define conditions appropriate for the introduction of DNA into L. bulgaricus.     

Transfection of Lactobacillus bulgaricus protoplasts by bacteriophage DNA.

A protoplast transfection system has been developed for Lactobacillus bulgaricus. The procedure involves a polyethylene glycol-mediated fusion of bacteriophage DNA encapsulated in liposomes into mutanolysin-treated cells. With L. bulgaricus B004 and DNA isolated from the phage phi c5004, transfection reached a maximum when at least 95% of the cells were osmotically fragile. The incorporation of phage DNA into liposomes was essential; no transfectants were detected in the absence of liposomes. The largest number of transfectants was observed after longer periods (20 min) of fusion of mutanolysin-treated cells and liposomes with polyethylene glycol. The maximum efficiency of 5 x 10(7) PFU/microgram of DNA was reached after a 24-h incubation in growth media prior to plating transfected cells in an agar overlay to detect the appearance of plaques. A minimum of 4 h of incubation in growth medium after fusion was required to detect the production and release of virions. The possibility that the high frequencies observed were due to bursting of transfected cells and subsequent infection of additional cells was found not to be a factor. The number of transfectants observed was directly proportional to the quantity of DNA added. These results define conditions appropriate for the introduction of DNA into L. bulgaricus.     

High-frequency transfection of mouse FM3A mammary carcinoma cells in suspension culture with plasmid DNA

High-frequency transfection of mouse FM3A cells, grown in suspension, with plasmid pSV2neo DNA was achieved by incubation of the cells with DNA plus polybrene for 6 h followed by an osmotic shock with a hypertonic NaCl solution. When incubated for 20 min at 34 degrees C, FM3A cells showed resistance to the osmolarity change from 0.1 to 9.0% NaCl in the medium. Within this concentration range, 5-7% gave the highest efficiency of transfection. Both linear and circular forms of plasmid DNA produced transformants with equal efficiency. This method was simple, reproducible, and carrier DNA was not required. The efficiency was about 100 times higher than that of the method with DNA-calcium phosphate precipitates. Transformed cells were stable and different numbers of plasmid DNA copies were detected.