Electroporation of primary neural cultures: a simple method for directed gene transfer in vitro

Gene transfer into cells of the nervous system is an important method to analyze tissue-specific gene functions. Although highest transfection efficiencies are generally obtained by viral gene transfer, non-viral methods are attractive because they are less labor intensive and more suitable for high throughput screening approaches. Here we describe an approach for electroporation-based gene transfer into primary neural cells isolated from dissociated murine cerebella. Using GFP as reporter molecule, we show that electroporation allows for efficient gene transfer into embryonic and postnatal neural cells under highly controlled experimental conditions. Furthermore we show that adaptation of electroporation parameters allowed for the preferential transfection of subsets of neural cells within the mixed primary culture. Using electroporation settings of high voltage and low capacitance (500 V/50 microF) we achieved a transfection efficiency of about 10% of small neural cells which were identified as granule cells by the expression of the granule cell-specific marker NeuN. At electroporation settings of 220 V/975 microF, large and stellate-shaped cells that comprised about 10% of the GFAP-positive population of astrocytes were preferentially transfected. We conclude that electroporation of primary neural cells can be used to target gene transfer to subsets of neural cells.     

Retina-specific gene expression and improved DNA transfection in WERI-Rb1 retinoblastoma cells

We have studied retina-specific gene expression and gene promoter activity in WERI-Rb1 retinoblastoma cells. In general, the expression of endogenous genes matched the efficiency of promoter activity of the transfected gene: interphotoreceptor retinoid binding protein and phosphodiesterase-beta mRNAs and reporter activities were readily detected while other retina-specific messages were at or below the detection limit in WERI-Rb1 cells. Phosphodiesterase-beta promoter appeared active in all six cell lines tested. The viral SV40 promoter is very weak in WERI-Rb1 cells, which has implications for its use in gene constructs targeted to the photoreceptors. Our results also show that polyethyleneimine 25 is an efficient and simple carrier for DNA. The optimized transfection conditions permit the use of 24-well plates and low amounts of DNA for improved analysis of promoter activities, as compared to previous studies. Our results are expected to facilitate further research on retina-specific gene expression.     

Quantitation of transient gene expression after electroporation.

The combination of a photometric reporter-gene assay, with transfection by electroporation, is potentially a rapid and sensitive tool for the study of genetic regulatory elements in many types of cells. We have found that the sensitivity, accuracy, and reproducibility of the technique is greatly improved by the inclusion of appropriately chosen carrier DNA as the primary DNA species present during electroporation. By using high levels of carrier, the activities of constructs of differing sizes can be quantitatively compared, active constructs can be assayed with sub-microgram amounts of plasmid, and the activities of the constructs are linear over a wide concentration of DNA. In addition, the activity of miniprep DNA can be screened without purification on CsCl gradients giving activities equal to CsCl-purified DNA. This is extremely useful when doing preliminary screening of large numbers of constructs for promoter or enhancer activities. We report the results of testing various types of DNA as carrier, and the parameters for optimizing its use.     

DNA transfection of Escherichia coli by electroporation

Electroporation was applied to transfection and transformation of Escherichia coli. Efficient transfer of DNA was achieved by a single voltage pulse at 2.5 kV (initial electric field strength = 6.25 kV/cm), with a 25 microF capacitor. As the recipient for transfecting DNA in the electroporation, spheroplasts, EDTA-treated cells and osmotically shocked bacteria were inferior to intact E. coli. Various parameters affecting the transfection efficiency were defined including growth phase of recipient cells, concentrations of DNA and cells, temperature and additions. In most strains tested, electroporation was far more efficient than Ca2+-dependent transfection (transformation). Various aspects of the electroporation-mediated DNA uptake are discussed.     

Efficient transfection and expression of heterologous genes in PC12 cells

The PC12 pheochromocytoma cell line has been a favorite model system for cell and neurobiologists, but has proven relatively refractory to standard DNA transfection methods. We have found that the cationic lipid "lipofectin" provides a simple, gentle, and nontoxic procedure that vastly improves transfection efficiencies in PC12 cells. Transient expression of chloramphenicol acetyl transferase (CAT) driven by a Rous sarcoma virus long terminal repeat (LTR) is much more efficient using lipofectin when compared with calcium phosphate as a transfection procedure. Additionally, transient transfection of nerve growth factor (NGF)-differentiated PC12 cells proceeds with equal efficiency relative to naive, uninduced cells. Using the lipofectin procedure, the frequency of stable transfection is 100-fold higher than that reported with standard calcium phosphate precipitation protocols. To examine the effectiveness of different promoters for efficient expression of heterologous DNA in PC12 cells, three different promoter-bearing constructs were utilized. Each construct contains a different promoter sequence upstream from a chicken calsequestrin cDNA. A human cytomegalovirus (CMV) immediate early promoter construct produced the highest level of expression, followed by a human beta-actin promoter construct. Expression from a mouse Moloney sarcoma virus LTR construct could not be detected. These results overcome the previous transfection problems of low efficiency and low viability that have plagued many PC12 cell investigations.     

Electroporation of lymphoid cells: factors affecting the efficiency of transfection

We have increased the efficiency of electroporation of lymphoid cells over fifty fold by optimising several biological and electrical parameters. Under optimised conditions, the electroporation efficiency was comparable to that reported for other cell types. Actively dividing cells were crucial for high transient transfection signal. The two most important electrical parameters were high capacitance (960 microF) and moderate decay constants in the range of 10-15 ms. The optimal field strength depended on the cell line, but was in the range 0.6-1 kV/cm. Administering the pulse in medium lacking serum gave higher efficiency than when isotonic salt solution was used and the transfection signal was depressed if cells and DNA were allowed to incubate for several minutes either before or after the pulse. Electroporation was carried out at room temperature and there was no advantage in using low temperatures (0-4 degrees C). When electroporated cells were grown in conditioned medium, the signal was enhanced about two fold depending on the source of the conditioned medium.     

Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood

Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34(+) hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34(+) hematopoietic cells were separated on immunomagnetic MiniMACS columns. Pure population of CD34(+) cells was incubated in a serum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of various pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 microF, 550 V/cm, and 10 microg of DNA per 500 microl. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capacitance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34(+) hematopoietic cells derived from human umbilical cord blood.     

Activation of CMV promoter-controlled glycosyltransferase and β -galactosidase glycogenes by butyrate, tricostatin A, and 5-Aza-2′-deoxycytidine

Cytomegalovirus (CMV) immediate early promoter is a powerful promoter frequently used for driving the expression of transgenes in mammalian cells. However, this promoter gradually becomes silenced in stably transfected cells. We employed Chinese Hamster Ovary (CHO) and human pancreatic cancer (Panc 1) cells stably tansfected with three glycogenes driven by a CMV promoter to study the activation of silenced glycogenes. We found that butyrate, tricostatin A (TSA), and 5-aza-2-deoxycytidine (5-Aza-dC) can activate these CMV-driven glycogenes. The increase in mRNA and protein of a glycogene occurred 8–10 h after butyrate treatment, suggesting an indirect effect of butyrate in the activation of the transgene. The enhanced expression of the trangenes by butyrate and TSA, known inhibitors of histone deacetylase, was independent of the transgene or cell type. However, the transgene can be activated by these two agents in only a fraction of the cells derived from a single clone, suggesting that inactivation of histone deacetylase can only partially explain silencing of the transgenes. Combination treatment of one or both agents with 5-Aza-dC, a known inhibitor of DNA methylase, resulted in a synergistic activation of the transgene, suggesting a cross-talk between histone acetylation and DNA demethylation. Understanding the mechanisms of the inactivation and reactivation of CMV promoter-controlled transgenes should help develop an effective strategy to fully activate the CMV promoter-controlled therapeutic genes silenced by the host cells. Published in 2005.     

Electroporation optimization to deliver plasmid DNA into dental follicle cells

Electroporation is a simple and versatile approach for DNA transfer but needs to be optimized for specific cells. We conducted square wave electroporation experiments for rat dental follicle cells under various conditions. These experiments indicated that the optimal electroporation electric field strength was 375 V/cm, and that plasmid concentrations greater than 0.18 μg/μL were required to achieve high transfection efficiency. BSA or fetal bovine serum in the pulsing buffer significantly improved cell survival and increased the number of transfected cells. The optimal pulsing duration was in the range of 45–120 ms at 375 V/cm. This electroporation protocol can be used to deliver DNA into dental follicle cells to study the roles of candidate genes in regulating tooth eruption. This is the first report showing the transfection of dental follicle cells using electroporation. The parameters determined in this study are likely to be applied to transfection of other fibroblast cells.     

Optimization of genetic constructs for high-level expression of the darbepoetin gene in mammalian cells

Genetic constructs were designed in order to optimize darbepoetin production in CHO cells. They are characterized by a higher level of structural optimization of the darbepoetin gene, a higher gene dose, and the selection of promoter elements that have never been used before for this purpose. A transient transfection of CHO cells by the obtained constructs was performed. It was shown that each of the variable factors in the constructs influenced darbepoetin gene expression. A construct containing a doubled dose of the darbepoetin synthetic gene with optimized codon composition under the control of the CMV-EF1α chimerical promoter was proved to be the most efficient.     

Generation of linear expression constructs by one-step PCR with vaccinia DNA topoisomerase I

Linear expression constructs can facilitate gene function studies. We describe a method to generate linear expression constructs for mammalian cells by one-step polymerase chain reaction (PCR) with vaccinia DNA topoisomerase I (TOPO). Cytomegalovirus (CMV) 5′ promoter, the gene of interest, and V5 bovine growth hormone (BGH) polyA 3′ terminator elements were PCR-amplified with target-specific primers containing vaccinia DNA TOPO-specific sequence and complementary sequence to each other. We amplified specific and complementary sequences. These three elements were directionally joined with vaccinia TOPO. The joined products were then directly transfected into Chinese hamster ovary cells. Compared with the transfection of supercoiled plasmids, comparable expression signals were obtained for green fluorescent protein, chloramphenicol acetyltransferase, and β-galactosidase proteins using Western blots. This is a quick and efficient method to generate linear expression construct. Unlike Invitrogen TOPO Tools, our method avoided the secondary round of PCR and more rapidly yielded correct joining products. This method can be easily used in the function test of uncharacterized open reading frames.     

Expression of the chimeric IgE gene in cell culture and in various mouse tissues

Transient expression of recombinant gene constructs is now more widely used in gene therapy as well as in DNA vaccination. In this study, the ability of one and the same genetic construct to drive gene expression both in cell culture and in tissues of the whole organism was demonstrated. Chinese hamster ovarian cells (CHO) were transfected in vitro with plasmids bearing the genes for chimeric IgE (mouse/human) antibodies under control of the human cytomegalovirus (hCMV) promoter. Secretion of recombinant IgE antibodies by transfected cells reached 60% of the intracellular concentration of antibodies. The same gene constructs were introduced into various mouse tissues using ballistic transfection in vivo. The IgE content in blood after transfection of cartilage was found to be several times lower than after transfection of the liver, spleen, or foot pad. At the same time, the content of antibodies to the xenogenous determinants of IgE was essentially independent of the tissue type. These data can be employed in selecting conditions for genetic immunization and gene therapy.     

Inactivation of lacZ gene expression by UV light and bound DNA photolyase implies formation of extended complexes in the genomes of specific Escherichia coli strains

Summary In Escherichia coli strains WU and CS101, UV inactivation of lacZ gene expression is more effective when the cells contain amplified DNA photolyase, and flash photoreactivation (fPR) after 15 min of metabolism does not reverse inactivation by the photolyasedimer complexes. In other strains, also studied with or without amplified DNA photolyase, there is no differential UV inactivation and fPR reverses inactivation by the complexes regardless of continued metabolism. The irreparable condition in strain WU is not due to dysfunction of photolyase: during post-UV metabolism, fPR still restores viability and dimers are removed from the region of the lac operon. When the wild-type lac promoter is repalced by the UV5 promoter, making expression insensitive to relaxed supercoiling and catabolite repression, inactivation by dimers alone becomes more resistant, i.e. requires higher fluences, but inactivation in WU and CS101 is still exceptionally sensitive to photolyasedimer complexes. This indicates that dimers external to the wild-type lac operon may inhibit expression by altering supercoiling but that complexes must involve some other mechanism for their special effect in WU and CS101. The exceptionally efficient inactivation and irreparable condition are consistent with the idea that, in two specific laboratory strains, photolyase bound to dimers at a considerable distance from the lac operon may initiate an aggregation of DNA with other cellular molecules that extends to, and inactivates expression from, the operon.     

Upper limit for DNA packaging by Bacillus subtilis bacteriophage phi 105: isolation of phage deletion mutants by induction of oversized prophages

Summary We have determined the upper size limit for DNA packaging in Bacillus subtilis bacteriophage 105 by examining the plaque-forming and transducing capabilities of lysates made from strains containing prophages of various sizes. The upper size limit for efficient packaging of the phage genome appears to be about 40.2 kb, which is about 1 kb larger than the wild-type genome. This places an upper limit of about 5 kb on the size of insertions that can be accommodated in 105 transfection cloning vectors, such as 105J27. Induction of prophages that exceed the upper limit, followed by selection for plaque formation or transduction, provides a powerful means of isolating phage deletion mutants. A comparison of the location of each deletion with the resultant phenotype has enabled us to identify non-essential regions of the phage genome, and regions that are required for tail biosynthesis and for host cell lysis.     

Electroporation and expression of the broad host-range plasmid pRK2501 in Azotobacter vinelandii.

Azotobacter vinelandii cells were transformed via high-voltage electroporation, with the broad host-range plasmid pRK2501. The number of transformants was dependent on the applied voltage, capacitance, and recovery procedure after electroporation. For example, Log, 4.44 transformants microgram-1 DNA were recovered in the A. vinelandii cell suspension electroporated at 1500 V and 25 microF capacitance (time constant 29.0 ms) and recovered on LB agar amended with 0.5 microgram/ml-1 kanamycin (pRK2501 encodes for both kanamycin and tetracycline resistance). Electroporation at 2500 V and capacitance settings of 25 and 3 microF did not produce any transformants. Cell survival was also poor at high voltages. A. vinelandii transformants were not recovered on N-free agar medium. In addition, no viable cells were recovered on N-free agar after electroporation at 2500 V, 25 microF; 2500 V, 3 microF; and 1500 V, 25 microF. Electroporation may be a useful method to genetically transform Azotobacter species for use in physiological and/or genetic studies.