Delivering siRNAs to Difficult Cell Types
Electroporation of Primary, Neuronal and Other Hard-to-transfect Cells
Dmitriy Ovcharenko, Rich Jarvis, Kevin Kelnar, David Brown, Ambion, Inc.  

RNA interference (RNAi) is a powerful experimental tool for reducing the expression of specific genes. It is used routinely for gene function analysis, target validation, and gene discovery. RNAi is also exploited to create new gene-specific therapeutics. Scientists performing RNAi experiments in mammalian systems must deliver siRNAs into cells where the siRNAs guide the RNA-induced silencing complex (RISC) to target mRNA for cleavage. Lipid-based transfection reagents are typically used for siRNA delivery in immortalized cell lines. However these reagents tend to be inefficient for siRNA delivery to most primary and neuronal cell types and to cells grown in suspension. Here we demonstrate the use of electroporation to deliver siRNAs into primary cells and other hard-to-transfect cell types. Ambion's siPORT™ siRNA Electroporation Buffer can be used with commonly available electroporators to ensure highly efficient siRNA delivery into cells while maintaining high levels of cell viability.

An Alternative to Lipid-based Transfection
Mammalian cells can be successfully loaded with exogenous siRNA when the correct method and matrix of transfection conditions are employed. Chemical transfection (e.g. using lipid-based reagents) is used routinely to deliver siRNAs into immortalized cells. Unfortunately, efficient transfer of siRNAs into primary cells by chemical transfection is restricted to only a few cell types [1]. Since primary cells are more similar to their in vivo counterparts than are immortalized cells, they serve as an important model system for in vivo applications. Better delivery methods for these cell types are needed.

As an alternative to chemical transfection, Ambion has investigated electroporation of primary cells and hard-to-transfect cell types. Electroporation involves applying an electric field pulse to induce the formation of microscopic pores in the cell membrane which allow molecules, ions, and water to traverse the membrane [2]. Under specific pulse conditions, the pores reseal and the "electroporated" cells recover and resume growth. A distinct advantage of electroporation over chemical methods is that it is not dependent on cell division, and RNAi-induced reduction in gene expression can be detected just a few hours after nucleic acid delivery.

Most existing electroporation protocols were developed to deliver plasmid DNA to the cell nucleus [3, 4]. These protocols often result in high cell mortality [5, 6, 7]. For transient RNAi experiments, it is important that cell trauma from electroporation be minimized to reduce any possible effects on gene expression patterns. Since siRNAs need only be delivered to the cytoplasm, milder electroporation conditions can be used that minimize cellular mortality and trauma while ensuring highly efficient siRNA delivery. In these experiments, electroporation was initially used to deliver Cy™3-labeled siRNA targeting GAPDH into human primary mesenchymal stem cells (hMSC) and rat neuronal pheochromocytoma (PC-12) cells. 24 hours after electroporation almost every cell contained detectable amounts of Cy3-labeled siRNA (Figure 1). To confirm that the siRNAs were functional, GAPDH expression levels were measured in the hMSC cells (Figure 2). While this experiment resulted in efficient delivery of siRNA, it also resulted in low cell viability. Next we developed an electroporation buffer and protocol to significantly increase cell viability after electroporation.

Figure 1. Electroporation of Primary Cells and Hard-to-transfect Neuronal Cells. A Cy™3-labeled GAPDH siRNA (1.5 µg) was added to primary Human Mesenchymal Stem Cells (hMSC) or rat neuronal pheochromocytoma (PC-12) cells and electroporated (75 µl) using hMSC-specific or PC-12-specific parameters. Cells were fixed 24 hours after electroporation, stained with DAPI (blue), and analyzed by fluorescence microscopy (Cy3-fluorescence; red).

Figure 2. Silencing Efficiency Depends on Amount of siRNA per Electroporation. Primary human mesenchymal stem cells (hMSC) were electroporated with various amounts of siRNA targeting GAPDH (0.1-3.0 µg) or a scrambled negative control siRNA. 24 hours post-transfection, cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels (Normalization control). mRNA remaining for GAPDH was calculated as a percentage of GAPDH mRNA detected in cells transfected with the negative control siRNA.

Electroporation Pulse Buffer and Electropulse Generators
Ambion's siPORT siRNA Electroporation Buffer is a low-conductivity buffer designed to emulate the natural composition of the cytoplasm. It enables delivery of siRNA into primary, neuronal, and other hard-to-transfect cell types, promoting siRNA-mediated knockdown of gene expression, while retaining high cell viability. Ambion's siPORT siRNA Electroporation Buffer works by facilitating rapid pore resealing. Using the siPORT siRNA Electroporation Buffer with square wave-type pulses, we were able to transfect hMSC cells, normal human dermal fibroblasts-neonatal (NHDF-Neo cells), normal human umbilical vein endothelial cells (HUVEC), Human acute T-cells (Jurkat) and rat neuronal PC-12 cells with high efficiencies (75.0-99.9%) and high cell viability (65-95%), as seen in Figure 3.

Figure 3. Silencing and Cell Viability after Electroporation. Successful gene silencing and high cell viability was achieved in 3 primary cell types: human mesenchymal stem cells (hMSC), normal human dermal fibroblasts-neonatal (NHDF-Neo), normal human umbilical vein endothelial cells (HUVEC) and 2 hard-to-transfect cell types: Jurkat and PC-12 (rat pheochromocytoma) cells. siRNA targeting GAPDH or scrambled negative control siRNA (1.5 µg) were electroporated. 24 hours post-transfection, the cells were harvested and analyzed by real-time RT-PCR for target mRNA levels. 18S rRNA levels were used to normalize GAPDH expression. Remaining mRNA % was calculated as a percentage of mRNA compared with the PC-12 negative control siRNA.

Electroporators
Transfection using the siPORT siRNA Electroporation Buffer was performed with two different electro-pulse generators: Gene Pulser Xcell™ (Bio-Rad) and ECM 830 (BTX). Both electroporators performed approximately equivalently when the same electroporation parameters were used, resulting in equivalent levels of siRNA uptake by several different cell types and similar reduction of gene expression (variability <10%; data not shown). The Gene Pulser Xcell System performed very consistently without sample-to-sample electropulse fluctuations across multiple experiments. The ECM 830 electropulse generator instrument performed with 5-10% sample-to-sample electropulse fluctuations across multiple experiments.

Optimizing siRNA Electroporation Parameters
Experiments revealed that different primary cell types require different electroporation parameters. For example, in a standard 1 mm electroporation cuvette, HUVEC cells require 1 electropulse, 150 µS, and 250 Volts; whereas primary NHDF-Neo cells require 2 pulses, 70 µS, and 900 Volts. Therefore, as with any transfection, it is important to optimize some critical protocol parameters to ensure maximum delivery of siRNA by electroporation. Transfection efficiency is largely affected by the concentration of siRNA, and can be modulated by varying the siRNA amount within a limited range. To demonstrate the relationship between gene silencing and siRNA concentration, hMSC cells were electroporated with five different concentrations of GAPDH siRNA or scrambled negative control siRNA. Real-time RT-PCR revealed a dose dependent reduction in GAPDH mRNA levels (Figure 2). In these experiments and others 0.5-2.5 µg siRNA (in a 75 µl electroporation volume) was found to be most effective.

Varying the number of pulses is overall the most influential parameter for achieving maximum reduction of target mRNA levels. For most mammalian cell types one pulse is usually sufficient to achieve at least 70% reduction in target gene expression. In general more pulses resulted in better siRNA uptake but increased cell mortality, thus the two must be balanced. Figure 4 shows the effect that varying pulses can have when a GAPDH siRNA or a control scrambled siRNA was electroporated into NHDF-Neo primary cells.

Figure 4. GAPDH Silencing and Cell Viability vs. Number of Electroporation Pulses in Primary Cells. siRNA targeting GAPDH or a scrambled sequence (1.5 µg) was electroporated into NHDF-Neo primary cells using a varying number of electroporation pulses. 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 GAPDH mRNA levels. Percent remaining gene expression was calculated as a percentage of gene expression compared with the negative control siRNA.

Summary
Electroporation with Ambion's siPORT siRNA Electroporation Buffer and optimized conditions provides a highly efficient method for transfecting primary and hard-to-transfect cells with siRNAs. This was demonstrated here using five different cell types. The results show that transfection via electroporation provides an efficient nonviral method to induce RNAi in cells that are resistant to siRNA delivery using chemical transfection agents. However, conditions vary with cell type and it is important to optimize siRNA concentration and pulse number to achieve optimal silencing. Delivery of siRNA to primary cell types, a setting where genetic manipulations have traditionally proved difficult, will be a valuable research tool in various applications including gene function analysis, target validation, gene discovery, and even development of gene-specific siRNA-based therapeutics.

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