Dmitriy Ovcharenko (Research Associate II, Ambion, Inc.)
Gene silencing using small interfering RNAs (siRNAs) has become a powerful method for studying gene function. Human primary cells are often desired for such experiments because they are more similar to their in vivo counterparts than are immortalized cells. The use of siRNAs in human primary cells continues to accelerate applications such as target validation, gene discovery, and gene therapeutic approaches that could lead to powerful new gene-specific siRNA-based therapeutics.
Chemical transfection is the standard method for introducing siRNA into immortalized cells. Unfortunately, efficient transfer of siRNAs into primary cells by chemical transfection has so far been restricted to a few cell types only. This is illustrated in Figure 1, below. Primary cells tend to be more difficult to transfect chemically in general, which has limited their use for siRNA experiments. Here we address the use of electroporation as an alternative for efficient delivery of siRNAs into primary cell types.
Chemical Transfection: Inefficient siRNA Delivery in Primary Cells
To show that chemical transfection is only useful as an siRNA delivery method for only certain types of primary cells, an siRNA targeting GAPDH was transfected into normal human umbilical vein endothelial cells (HUVEC) and normal human epidermal keratinocytes (NHEK) using a panel of chemical transfection reagents. Cells were harvested 48 hours post transfection and analyzed for GAPDH expression using real-time RT-PCR.
In HUVEC cells, use of siPORT Amine transfection reagent (Ambion) elicited a decrease in GAPDH gene expression by more than 85%. Reduction of GAPDH gene expression by >50% was accomplished in HUVEC cells using several siRNA transfection agents from other manufacturers (Figure 1, Panel A). In contrast, none of the transfection agents were capable of delivering enough GAPDH siRNA to NHEK cells to reduce GAPDH expression by more than 35% (Figure 1, Panel B). This example was chosen to emphasize that primary cells can vary widely in their ability to be efficiently chemically transfected, even when a wide variety of transfection agents and protocols are used.
Figure 1. Reduction of GAPDH Gene Expression in HUVEC and NHEK Cells Using Various Transfection Agents. A chemically synthesized siRNA targeting GAPDH and a scrambled siRNA sequence were individually transfected into HUVEC cells (Panel A) or NHEK cells (Panel B) using various commercially available transfection agents. Manufacturer standard protocols were used for all transfections. 48 hours post-transfection, the cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. 18S rRNA levels were used to normalize the GAPDH expression data. Percent expression was calculated as a percentage of gene expression compared to the negative control siRNA. Duplicates were performed for each sample.
An alternative to chemical transfection-mediated nucleic acid delivery is electroporation. This method involves applying an electric field pulse to induce the formation of microscopic pores (electropores) in the cell membrane. This allows molecules, ions, and water to traverse the membrane. Under specific pulse conditions, the electropores reseal and the "electroporated" cells recover and continue to grow. A distinct advantage of electroporation is that it is not dependent on cell division, and reduction in gene expression can be detected a few hours after nucleic acid delivery. This is important when working with primary cells since most divide relatively slowly if at all.
We used electroporation (Nucleofector, Amaxa) to deliver Cy3-labeled siRNA targeting GAPDH into HUVEC cells. A HUVEC-specific protocol (Amaxa) was used. We were pleasantly surprised to find that almost every cell had taken up detectable amounts of Cy3-labeled siRNA (Figure 2). The Nucleofector was then used to electroporate HUVEC, NHEK, and normal human dermal fibroblasts neonatal (NHDF-Neo) with five different concentrations of GAPDH siRNA. Real time RT-PCR revealed a dose dependent reduction in GAPDH expression in all three cell types. Some toxicity was observed at the highest siRNA dose tested (2.66 g), but there was a complete absence of non-specific effects at 1.33 g siRNA (Figure 3). To ex tend these results, validated siRNAs targeting CDK2, p53, and JAK1 were individually electroporated into the same set of primary cells. All three genes were efficiently silenced (Figure 4).
Figure 2. Electroporation of HUVEC Primary Cells. A Cy3-labeled GAPDH siRNA (1.33 g) was added to HUVEC primary cells and electroporated using the HUVEC-specific protocol (Amaxa) and Nucleofector machine (Amaxa). Cells were fixed 24 hours after electroporation and stained with DAPI (blue), Cy3 fluorescence (red).
Figure 3. Reduction of GAPDH Gene Expression in HUVEC, NHEK, and NHDF-neo Human Primary Cells. Different primary cell lines were electroporated using Amaxa Nucleofector technology. Varying amounts from 66 ng to 2.66 g of siRNA targeting GAPDH or 18S rRNA were used. 24 hours post-transfection, the cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. 18S rRNA levels were used to normalize the GAPDH data. Percent gene expression was calculated as a percentage of gene expression compared with the negative control siRNA. Duplicates were performed for each sample.
Figure 4. Validated siRNAs Elicit RNAi When Electroporated into Several Cell Types. Three siRNAs (1.33 g) targeting CDK2, p53, JAK1 and a scrambled sequence were electroporated into HUVEC, NHEK, NHDF-neo human primary cell lines. 24 hours post-transfection the cells were harvested and analyzed by real-time RT-PCR for gene expression levels. 18S rRNA levels were used to normalize the CDK2, p53, JAK1 data. Percent gene exp ression was calculated as a percentage of gene expression compared with the negative control siRNA. Duplicates were performed for each sample.
Our results demonstrate that electroporation provides an efficient non-viral method for delivering siRNAs in at least these three types of primary cells. In contrast to chemical transfection reagents, electroporation of a GAPDH siRNA reduced GAPDH expression by greater than 90% across several primary cell types, including NHEK cells, which are only poorly transfected with several common chemical transfection agents. The application of siRNA to primary cell types, a setting where genetic manipulations have traditionally proven difficult, will be a valuable tool in studies on target validation, gene discovery, and gene therapeutic approaches.
Note that it is common to see relative variations in data when conducting tissue culture experiments. Reproducibility can be achieved by rigorously following protocols.
Cy is a trademark of Amersham Biosciences.
siPORT Amine is manufactured for Ambion by Mirus.
TaqMan is a registered trademark of Applied Biosystems.