BackgroundThe delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected.ResultsStudies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells) and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts). With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific.ConclusionsIn general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival.

Little is known about the relationship between CF transmembrane conductance regulator (CFTR) gene expression and the corresponding transport of Cl. The phenotypic characteristics of polarized ΔF508 homozygote CF bronchial epithelial (CFBE41o-) cells were evaluated following transfection with episomal expression vector containing either full-length (6.2kb) wild type (wt) and (4.7kb) ΔF508CFTR cDNA. Forskolin-stimulated Cl secretion in two clones expressing the full-length wild type CFTR was assessed; clone c7-6.2wt gave 13.4±2.5 µA/cm² and clone c10-6.2wt showed 41.3±25.3 µA/cm². Another clone (c4-4.7ΔF) complemented with the ΔF508 CFTR cDNA showed high and stable expression of vector-derived ΔF508 CFTR mRNA and a small cAMP-stimulated Cl current (4.7±0.7 µA/cm²) indicating ΔF508CFTR trafficking to the plasma membrane at physiological temperatures. Vector-driven CFTR mRNA levels were 5-fold (c7-6.2wt), 14-fold (c10-6.2wt), and 27-fold (c7-4.7ΔF) higher than observed in normal bronchial epithelial cells (16HBE14o-) endogenously expressing wtCFTR. Assessment of CFTR mRNA levels and CFTR function showed that cAMP-stimulated CFTR Cl currents were 33%, 167% and 24%, respectively, of those in 16HBE14o- cells. The data suggest that transgene expression needs to be significantly higher than endogenously expressed CFTR to restore functional wtCFTR Cl transport to levels sufficient to reverse CF pathology.

Thermal denaturation experiments have established that an oligonucleotide incorporating the artificial nucleobase S, does form a stable triplex with a double stranded DNA which exhibits a pyrimidine interruption within the oligopurine sequence.

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