Comparative transfection of DNA into primary and transformed mammalian cells from different lineages

Abstract

Background: The 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.

Results: Studies 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.

Conclusions: In 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.

Figure 1

The transfection efficiency obtained 48 hours after nucleofection of 10⁶ cells with 2 μg of pmaxGFP plasmid. The cells are described in Table 1. The error bars represent the standard error of the mean (SEM), with n = 3.

Figure 2

Comparison of the transfection efficacy of pmaxGFP with chemical reagents (Effectene, Lipofectamine 2000, Lipofectamine Plus, and PEI) and nucleofection. The vol/wt ratios (μl reagent/μg DNA) for Effectene and Lipofectamine 2000 transfection and the (+/-) charge ratios (PEI nitrogen residues/DNA phosphates) for PEI transfection are indicated in parentheses. Transfection efficacy is indicated by the black bar, and the relative number of adherent cells in the transfected cells was compared to the number in nontransfected control cultures is indicated by the white bar for (A) pig fetal fibroblast (P16), (B) primary pig tracheal epithelial (PTE) cells, and (C) primary human tracheal epithelial (HTE) cells. The error bars reflect the standard error of the mean (SEM), with n = 3.