Optimal Transfection Conditions Research Articles

Effect of flanking matrix attachment regions on the expression of microinjected transgenes during preimplantation development of mouse embryos

Transient gene expression in mammalian cells is intensively used for the rapid generation of recombinant proteins for biochemical, biophysical and pre-clinical studies. Still, the principles behind DNA transfer to the cells and the cellular cascade of events that ultimately dictate protein expression levels are not fully understood. Using polyethylenimine (PEI) mediated transfection of HEK293-EBNA1 cells, we sought to determine the most critical parameters that drive and limit recombinant protein production. Our results showed that a maximum of 65,000 plasmid copies/cell can be recovered in total extracts at 1 day post-transfection. Analyses performed after cell sorting revealed equal amounts of plasmid DNA in GFP-positive and -negative populations. However, nuclear plasmid content was three-fold higher in GFP-positive cells (1850 copies) than in GFP-negative cells (550 copies). The fact that significant amounts of plasmid DNA are found in the nucleus of GFP-negative cells suggests that its transcriptional competency is impaired. Interestingly, transfecting cells using a wide range of plasmid quantities at the optimal DNA:PEI ratio did not significantly affect the number of expressing cells. Thus, it appears that successful transgene expression is more likely to depend on a cellular “competent” state than to the quantity of plasmid DNA delivered per cell. Moreover, Northern blot analysis and SEAP/GFP measurement following plasmid titration experiments showed that transcriptional and translational processes are operating near to saturation under optimal transfection conditions. Overall, our results suggest that events that regulate nuclear translocation of plasmid DNA and its transcriptional competency as well as translational/post-translational limitations represent major bottlenecks in the success of a PEI-mediated protein production.

Transient and stable transfection of Leishmania by particle bombardment

Transfection of trypanosomatids, particularly the transfection of their mitochondrion and different developmental stages, is still a limiting step in many experiments using recombinant DNA technology. Two types of trypanosomatid transfection protocols have been described so far. The standard protocol is based on electroporation first described by Bellofatto and Cross [1]. The second protocol is based on transfection using a homemade particle gun [2]. The biolistic method was first applied to plants [3], but is now routinely used for transfection of a wide variety of targets ranging from bacterial to mammalian cells [4–10] and it is the only method described so far to transfect organelles [10,11]. In trypanosomatids, the genetic organization of the mitochondrion shows several unusual features like a complex DNA network composed of thousands of catenated circular DNA molecules [12], the lack of encoded tRNAs [13] and the still enigmatic process of RNA editing [14]. However, the trypanosomatid mitochondrion is not yet accessible to recombinant DNA technology, and therefore the experimental in vivo analysis of the mentioned features is severely restricted. It is intended that the particle bombardment technique should be applied to transfect mitochondria of trypanosomatids. Since the optimal bombarding conditions for nuclear and organellar transfection of a given organism generally are identical [6,10,15,16] and as a first step, the factors influencing the nuclear transfection efficiency have been investigated. The ability of microprojectiles to deliver DNA into Leishmania cells was demonstrated either by the presence of transient b-glucuronidase (GUS) activity or by selection of stable transformants on plates following bombardment with the plasmid pX63NEO-GUS [17] using the DuPont Biolistic Abbre6iations: GUS, b-glucuronidase. * Corresponding author. Tel.: +41 31 6314342; fax: +41 31 6313383; e-mail beat.blum@ibc.unibe.ch

Primary Culture and Transfection of Epithelial Cells of Human Small Intestine

Background: So far, no techniques are available for primary culture and efficient transfection of human small-intestinal enterocytes, which would provide a valuable tool to investigate intestinal function. Methods: Human small-intestinal biopsy specimens were treated with collagenase and dispase. Resulting crypt units were cultured for several days. Using the intestinal epithelial cell lines Caco-2 and HT-29, we established optimal conditions for transfection of a control plasmid, which were then applied to primary cultured cells. Results: Cells growing out of crypt units formed monolayer-like sheets and proliferated for several days. Most of the cells could be stained with antibodies against epithelial markers. Among seven different transfection reagents tested, Lipofectamine was the most potent, with transfection efficiencies up to 25% for primary enterocytes. Conclusions: An easy technique was developed providing viable small-intestinal enterocytes that can be efficiently transfected.

Quantitation ofin VivoGene Delivery by Restriction Enzyme PCR Generated Polymorphism

The Multiple intestinal neoplasia (Min) mouse develops multiple polyps in the intestine, due to a heterozygous mutation of the Apc locus. Our laboratory has been introducing normal human adenomatous polyposis coli (APC) gene into the Min mouse through liposome enema to prevent or reverse polyp formation. We have quantitated the amount of normal human APC gene deliveredin vivoby a restriction enzyme site specific quantitative PCR. Adult Min and BALB/C mice were treated with lipofectant and human APC complementary DNA (cDNA) plasmid. Min colonic DNA was amplified using primers for Apc nucleotide 2524F (5′2524-TCTCGTTCTGAGAAAGACAGAAGCT) and 2679R (5″2679-TGATACTTCTTCCAAAGCTTTGGCTAT). Highlighted primer sequences were purposely different so as to generate twoHindIII restriction enzyme sites in the presence of normal mouse Apc (Apc+). Genomic DNA from untreated Min colonic epithelium revealed two bands: 144 bp for ApcMinand 123 bp for Apc+. BALB/C DNA was amplified using primers flanking a region within the APC gene containing aHindIII site on the human APC, which is absent in the murine APC (Apc). Min's DNA extracted 24 hr after treatment demonstrated a plasmid content of 3% due to a relative increase in the Apc+(123 bp) band. Six weeks of treatments increased delivery to 10%. APC gene therapy of colonic epithelium can be quantitatively measured through restriction enzyme quantitative PCR. Long-term treatment further increases gene delivery. PCR generated polymorphism is a reliable and reproducible technique to initially optimize transfection conditions and ultimately quantitate efficacy in anin vivogene delivery model.

Transfection ofActinomycesspp. by Genomic DNA of Bacteriophages from Human Dental Plaque

Bacteriophages that produced turbid or clear zones of lysis in strains ofActinomyceswere isolated from 22 of 124 samples of fresh human dental plaque. All human and nonhuman strains ofActinomyces viscosusorActinomyces naeslundiitested in this study were sensitive to infection by one or more of these phages. In contrast, none of theActinomyces odontolyticus, Actinomyces israelii,orActinomyces bovisstrains tested were susceptible. Results of restriction endonuclease analyses indicated that the genomes of these phages consisted of double-stranded DNA molecules ranging in size between 16 and 60 kbp. Sequence homology under hybridization conditions of high stringency was observed among a few of the isolated phages. A lysogenized isolate ofA. viscosusMG-1 was obtained following infection with a temperate phage, designated φ225. Results of Southern blot analyses indicated that φ225 replicated as a plasmid in the lysogenized strain. Genomic DNA from several lytic phages was used to establish conditions for transfection by electroporation of strains ofActinomycesspp. Efficiencies of DNA transfer ranged from 102to 105plaque-forming units per microgram of DNA were obtained under optimal transfection conditions. The results of these studies demonstrate that transfer of genetic information inActinomycesspp. can be achieved by transfection.

Pharmaceutical studies for gene therapy: expression of human Cu, Zn-superoxide dismutase gene transfected by lipofection in rat skin fibroblasts.

To evaluate whether lipofection using Lipofectin is suitable for delivering foreign genes into skin fibroblasts as target cells, we performed experiments using human superoxide dismutase (hSOD) and neomycin-resistance (Neo) genes as models in rat skin fibroblasts (FR and primary cells) in vitro. The amounts of DNA used in the lipofection procedure significantly affected the transfection efficiencies, and the optimal amounts were determined for all cells used. However, the efficiencies in rat skin fibroblasts were about 20-fold higher than that in rat lung epithelial-like cells (L2 cells). The differences in plasmid vectors (pRc/RSV-SOD and pRc/CMV-SOD) hardly affected the transfection efficiencies. The amounts of Lipofectin significantly affected the transfection efficiencies, and the optimal amounts were determined for both types of skin fibroblasts. However, cytotoxic effects in both skin fibroblasts were observed with high doses of Lipofectin. On the other hand, with optimal amounts of DNA and Lipofectin, the reporter gene (NeoT) introduced into cells was mainly integrated into the host cell chromosome. Western blot analysis showed the continuous expression of hSOD protein for at least 45 d in skin fibroblasts transfected with the expression plasmid for hSOD by Lipofectin under the optimal conditions, and the cellular SOD activity fluctuated in parallel with the expression of hSOD protein. Differences in the type of cells also affected the expression of hSOD. These results indicate that it is necessary to set up optimal conditions for transfection using Lipofectin for each cell type, and that transfection with Lipofectin under optimal conditions may be an efficient method for introduction of foreign genes into skin fibroblasts for use as a clinical delivery system of therapeutic protein.

A new cat reporter gene vector designed for rapid and efficient cloning of PCR products

A cat reporter gene plasmid was constructed, which can be used very efficiently to clone PCR-derived promotor and enhancer fragments from genomic DNA. The new vector system pEKOCAT combines the efficiency in cloning with the approved low background of the pBLCAT6 vector. Additionally, the plasmid pEKSVCAT was constructed including the SV40 early promoter/enhancer to efficiently drive the cat reporter gene in particular cell lines. It can be used to optimize transfection conditions and as an internal positive control.

Optimization of Calcium Phosphate Transfection for Bovine Chromaffin Cells: Relationship to Calcium Phosphate Precipitate Formation

Optimal conditions for formation of calcium phosphate-DNA precipitates and for chromaffin cell transfection by the calcium phosphate method were examined. A relationship was observed between turbidity of calcium phosphate solutions and the ability of calcium phosphate-DNA mixtures to give efficient transfection of bovine chromaffin cells. Under optimal conditions up to 35% of chromaffin cells in cultures transfected with plasmid DNA encoding human proenkephalin or Escherichia coli β-galactosidase expressed the respective proteins. Important factors for transfection were the pH (6.95) and buffer employed for calcium phosphate-DNA precipitate formation, the amount and type of DNA, and the absence of serum in the cultures. Additionally, phosphate and calcium concentrations in the culture medium during incubation of cells with DNA are critical. Optimal conditions for transfection of chromaffin cells were also useful for transfection of clonal BSC-40 cells, an African green monkey kidney cell line. These results suggest that the optimal conditions described here for chromaffin cells may have broad applicability to other cell types. In addition, the results suggest that it is possible to optimize the solutions used for transfection conditions by monitoring calcium phosphate formation.

A new approach to the cloning of genes encoding T-cell epitopes.

The molecular structure of antigens recognized exclusively by T cells, such as minor histocompatibility antigens and some antigens that provoke autoimmune responses, has proved difficult to determine. Recently, several antigens induced on tumor cells by mutagen treatment have been cloned by transfection of genomic DNA libraries into P1.HTR cells, screening for antigen expression using T-cell clones, and subsequent recovery of the integrated DNA by cosmid rescue. We have modified this technique and have stably transfected P1.HTR cell lines with polyoma T antigen, which allows episomal replication of the shuttle vector, pCDM8. Using pCDM8-CAT constructs, we have determined the frequency of transfection and plasmid copies taken up per cell under optimal transfection conditions. Using a pCDM8 construct which expresses the tumor-specific antigen, P91A (pCDM8-tum-), that is recognized by a T-cell clone, we have found that cells transfected with this antigen can be recognized by the T-cell clone when they are present at only 1%-3% of a mixed population. Progeny of a single cell transfected with pCDM8-tum-: pCDM8-CAT at proportions of 1:10, 1:25, and 1:50 are recognized by the T-cell clone. Furthermore, Hirt extracted plasmid DNA from transfectants expressing the tum- antigen can be amplified in bacteria, transfected back into P1.HTR recipients, and recognized by the T-cell clone. This approach should enable reasonably rapid screening of cDNA libraries for even relatively low abundance messages encoding, for example, minor histocompatibility and alloantigens, and allow their subsequent cloning.

Comparison of Methods for Transfection of Human Epidermal Keratinocytes

Several methods for DNA-mediated cell transfection were tested to determine the optimal conditions for transfection of human epidermal keratinocytes. The following methods were compared: electroporation, lipofection, Ca3(PO4)2 co-precipitation, DEAE-dextran, and polybrene-mediated transfection. The transfected DNA included human keratinocyte-specific promoter for keratin K14 as well as SV40 and RSV viral promoters. Enzyme assays and in situ staining were used to evaluate both quantitative and qualitative aspects of transfection, and both subconfluent and post-confluent, stratifying keratinocytes were examined. Lipofection, Ca3(PO4)2 co-precipitation, and polybrene methods transfect very efficiently, but lipofection is expensive and Ca++ in the co-precipitation procedure induces keratinocytes to differentiate. We have found that polybrene-mediated transfection followed by a 27% DMSO shock is optimal for introducing DNA into human epidermal keratinocytes.

Analysis of leaky viral translation termination codons in vivo by transient expression of improved ?-glucuronidase vectors

Plant RNA viruses commonly exploit leaky translation termination signals in order to express internal protein coding regions. As a first step to elucidate the mechanism(s) by which ribosomes bypass leaky stop codons in vivo, we have devised a system in which readthrough is coupled to the transient expression of beta-glucuronidase (GUS) in tobacco protoplasts. GUS vectors that contain the stop codons and surrounding nucleotides from the readthrough regions of several different RNA viruses were constructed and the plasmids were tested for the ability to direct transient GUS expression. These studies indicated that ribosomes bypass the leaky termination sites at efficiencies ranging from essentially 0 to ca. 5% depending upon the viral sequence. The results suggest that the efficiency of readthrough is determined by the sequence surrounding the stop codon. We describe improved GUS expression vectors and optimized transfection conditions which made it possible to assay low-level translational events.

Gold bullets fight cancer

Recently, particle bombardment has become increasingly popular as a transfection method, because of a reduced dependency on target cell characteristics. In this study, we evaluated in vitro gene transfer by particle bombardment.gWIZ luciferase and gWIZ green fluorescent protein (GFP) plasmids were used as reporter genes. Mammalian cell lines HEK 293, MCF7 and NIH/3T3 were used in the transfection experiments. Transfection was performed by bombardment of the cells with gene-coated gold particles using the Helios Gene Gun. The technology was assessed by analyzing gene expression and cell damage. Cell damage was evaluated by MTT assay.This technology resulted in efficient in vitro transfection, even in the cells which are difficult to transfect. The gene expression was dependent on the gene gun's helium pressure, the sizes of the gold particles, the amount of the particles and DNA loading, while cell viability was mostly dependent on helium pressure and amount of the gold particles.This technology was useful to transfection of cells. Optimal transfection conditions were determined to be between 75 and 100 psi of helium pressure, 1.0 to 1.6 μm gold particle size and 0.5 mg of gold particle amount with a loading ratio of 4 μg DNA/mg gold particles.These findings will be useful in the design of gene gun device, and bring further improvements to the in vitro and in vivo transfection studies including gene therapy and vaccination.