Human embryonic stem cells (hESC) are unique in their ability to continuously self-renew while maintaining the ability to differentiate into any cell in the adult body . These characteristics give hESC the potential to be useful in many different aspects of basic and clinical research, including use as an ex vivo source for cellular transplantation; production of cells for screening drug candidates and assessing toxicity; and as an in vitro system for modeling human development and disease. However, the difficulty in producing genetically modified hESC lines has hindered the development of some of the most promising applications of hESC research (reviewed in ). Without the ability to easily and efficiently produce genetically modified cell lines, tools such as reporter cell lines, differentiation strategies based on the expression of certain growth factors, and the isolation of specific populations based on marker expression, will continue to limit the applications of hESC research.
The most common strategy for introducing exogenous nucleic acids into traditional, cultured cells is chemical transfection. In this process, DNA is introduced into the cells via liposomes or polycationic polymers (reviewed in ). While this process is relatively inexpensive and the vectors used have very few limitations, there are two major disadvantages. The first is that while many different chemical transfection strategies have been attempted to genetically modify hESC, the resulting efficiencies have generally been very poor, often significantly less than 1% (reviewed in ). An additional problem is that even when the exogenous DNA is successfully introduced into the cell, integration into the genome is poor, and when it does occur in hESC, the exogenous gene is often silenced, making isolation of stable transfectant cell lines inefficient [5, 6]. The low efficiency of transfection combined with the inefficient integration of the construct into the genome makes the average efficiency for generating stable transfectant clones in hESC around 1 in 105 .
The bulk of successful genetic modification of hESC has been done with lentiviral vectors (reviewed in ). Lentiviral vectors are advantageous in several ways, most important is their ability to integrate into the cellular genome to produce stably expressing cell lines . Lentiviral vectors have been shown to transduce many cell types (including primary and non-dividing cells) at high efficiencies and have been successfully used to transduce hESC . Unfortunately, the use of these vectors has two major disadvantages. First, vector constructs larger than 11 kb package poorly, reducing the efficiency of transduction and effectively limiting the size of DNA that can be transferred . The other disadvantage is the introduction of insertional mutagenesis. Since these vectors integrate randomly, they have the potential to activate oncogenes or inactivate tumor suppressor genes in the transduced cells, therefore raising safety concerns for potential clinical applications .
A third strategy for obtaining genetically modified cells is electroporation. In this method, DNA enters cells after an electrical current induces pore formation in the plasma membrane. While a disadvantage of this technique is reduced cell survival, electroporation has been successful in hESC [11–13]. As with chemical transfections, electroporation is not limited with respect to the size of DNA elements that can be inserted into the cells. Moreover electroporation is highly versatile in that it can be used to introduce other biological molecules such as exogenous proteins, mRNA or siRNA [14–18]. However, in most applications, electroporation has been difficult to scale for high-throughput usage.
Here we demonstrate that high throughput electroporation with either of two systems, the Nucleofection® 96-well Shuttle® System (Lonza AG, Cologne, Germany) or the Neon System (Invitrogen, Carlsbad, CA, USA), can efficiently deliver exogenous DNA into H9 human ESC, and that the viability of the cells after transfection remains high. These systems were chosen because they might conceivably provide high-throughput applications--the Shuttle using a 96-well plate with well-by-well transfection and the Neon with convenient pipet tip-based transfection. We found that both instruments provide suitable transfections in hESC but that the Shuttle system had more consistent results for high throughput applications so we chose it for further investigation. To determine whether the methods developed will be generally suitable for hESC lines, we transfected several additional lines and found no significant differences in transfection efficiency or cell viability. We also demonstrate that siRNA oligonucleotides can be efficiently transfected to knock down protein expression.
As is the case with chemical transfections, DNA introduced by the Shuttle System is only rarely integrated into the genome. However, the high throughput capability of the Shuttle System allows the generation of large numbers of viable transfectants, increasing the chance that successful genomic integration can be recovered, as demonstrated by the generation of a stable cell line expressing red fluorescent protein (RFP). Although we made no attempt to directly compare promoters and antibiotic resistance markers, the process of generating stable lines allowed us to make some conclusions about the most effective promoter/resistance marker combinations. As reported by others, we also noticed that two of the most widely used viral drivers, CMV (cytomegalovirus immediate-early promoter) and SV40 (simian virus-40 early promoter/origin region) are less likely to confer stable expression in hESC [19, 20].
Our study demonstrates that electroporation can be used in a high-throughput manner to produce either transient or stable transfectants in hESC with good viability. This will to be valuable for a variety of studies using hESC.