Ambion Poster: Efficient Delivery of siRNA to Primary Cells and Hard-to-Transfect Cell Lines

Efficient Delivery of siRNA to Primary Cells and
Hard-to-Transfect Cell Lines

Dmitriy Ovcharenko, Kevin Kelnar, Rich Jarvis

Ambion, Inc., • 2130 Woodward, Austin, TX 78744 • Phone: 512-651-0200 • Email: rjarvis@ambion.com

Mammalian cells can be successfully loaded with exogenous siRNA routinely when the correct method and matrix of transfection conditions are employed. Small molecule delivery can be highly efficient and yield almost 100% transfection of cells in culture. There are numerous technologies available to deliver siRNA (lip ofection, transduction, electroporation, etc.), and designing effective transfection strategies for a given cell type usually involves testing an assortment of reagents and methods. Many immortalized cell lines can be successfully transfected with chemical transfection agents, while finite cell lines or freshly isolated primary cells are more refractory and often require the use of alternative approaches. In multiple immortalized cell lines, a newly developed lipid-based transfection agent provided high levels of transfection efficiency and subsequent gene silencing when complexes of very low concentrations of siRNA (0.03-10 nM) were added directly to cells at the time of seeding even in the presence of serum. The method required minimal optimization to achieve silencing of greater than 95% of target gene expression.

As an alternative to lipofection, electroporation can provide higher transfection efficiencies in primary and hard-to-transfect cell types. These cells are often desired for such experiments because they are more similar to their in vivo counterparts than are immortalized cells. This method involves applying an electric field pulse to induce the formation of microscopic pores (electropores) in the cell membrane allowing 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 just a few hours after nucleic acid delivery. The use of siRNAs in 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. Here we explore delivery techniques for maximizing siRNA-mediated RNAi in a subset of human primary cells. Historically, electroporation protocols used for the delivery of larger plasmid DNA frequently resulted in high cell mortality. We have found that the electroporation of siRNA allows the use of milder conditions that induce minimal cellular toxicity. Using electroporation buffers designed specifically for siRNA delivery, high cell viability in primary cells and hard-to-transfect mammalian cell types can be attained. With these buffers, electroporation efficiencies of up to 95% are attainable while retaining over 90% cell viability in primary cell cultures.

Reagent-based Delivery of siRNA In Vitro

Figure 1. Potency of a New Transfection Agent Complexed with Various Amounts of siRNA. GAPDH siRNA or a scrambled control was complexed with a new transfection agent and added to empty wells of a 24 well dish. Sufficient amounts of siRNA were added to the wells to produce a final concentration range of 0.03, 0.10, 0.30, 1.0, 3.0, and 10 nM siRNA upon the addition of 500 µl cell suspension. HeLa cells were added to the wells and cultured for 48 hours in complete media containing serum. Following transfection, target gene expression was quantified by real time RT-PCR. Relative reduction in mRNA levels is expressed as a percentage of cells transfected with a scrambled control siRNA. Samples were normalized by measuring 18S rRNA levels by real time RT-PCR.

Figure 2. Effectiveness of a New Transfection Agent in Multiple Cell Lines. GAPDH siRNA or a scrambled control was complexed with a new transfection agent and added to empty wells of a 24 well dish. Sufficient amounts of siRNA were added to the wells to produce a 10-30 nM final concentration upon the addition of 500 µl cell suspension. A549, HeLa, MCF-7, BJ, and SKNAS cells were added to the wells and cultured for 48 hours in complete media containing serum. Following transfection, target gene expression was quantified by real time RT-PCR. Relative reduction in mRNA levels is expressed as a percentage of cells transfected with a scrambled control siRNA. Samples were normalized by measuring 18S rRNA levels in the various samples using real-time RT-PCR.

Delivery of siRNA by Electroporation:
An Effective Non-viral Method for Gene Silencing in Primary Cells

Figure 3. Efficiency of siRNA Delivery into Primary Cells by Electroporation. Cy3-labeled siRNA (1.33 µg) was electroporated into human primary cells according to an optimized protocol (data not shown). After 24 hours, cell viability was determined and cells were subsequently fixed and stained with DAPI (blue). Cy3 fluorescence (red) was analyzed by fluorescence microscopy. Data show that normal human umbilical vein endothelial cells (HUVEC) gave 99% transfection efficiency and 50% cell viability (Panel A) and normal human mesenchymal stem cells (hMSC) gave 99% transfection efficiency and 90% cell viability (Panel B).

Optimization of Critical Electroporation Parameters

Figure 4. Effects of Electroporation Field Strength and Pulse Length. PC-12 rat pheochromocytoma cells were electroporated using a Cyto Pulse PA-4000S - Advanced PulseAgile® Rectangular Wave system and Cyto Pulse Low Conductivity electroporation media. Varying wave forms were tested to determine the optimal conditions for the delivery of 140 ng GAPDH siRNA. 48 hours post-transfection, cells were harvested and analyzed by Northern blot analysis and bands were quantitated by densitometry. Percent gene expression was calculated as a percent of non-electroporated controls. Duplicates were performed for each sample. Cell viability was carried out using the using the Guava PCA and ViaCount™ Assay. Cell viability was calculated as a percent of viable cells before electroporation and two hours post-electroporation (Panel A). Images show fluorescence microscopy analysis of Cy3-labeled siRNA (below) or unlabeled control siRNA (above) uptake by PC12 cells 24 hours after delivery by electroporation (Panel B).

Figure 5. Reduction of GAPDH Gene Expression in NHDF-neo Primary Cells and Cell Viability over Varying Number of Electroporation Pulses. 1.5 µg of siRNA targeting GAPDH and scrambled sequence were electroporated into NHDF-neo primary cells using different 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 expression. Percent gene expression was calculated as a percentage of gene expression compared with the scrambled control siRNA. Electroporations were performed in duplicates.

Figure 6. Reduction of GAPDH Gene Expression in HUVEC, NHEK, NHDF-neo Human Primary Cells over Varying Amounts of siRNA. Multiple primary cell types were electroporated with varying amounts of siRNA (from 66 ng to 2.66 µg) targeting GAPDH or scrambled sequence. 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 GAPDH expression. Percent gene expression was calculated as a percentage of gene expression compared with the negative control siRNA. Electroporations were performed in duplicates.

Silencing of Multiple Gene Targets

Figure 7. Validated siRNAs Elicit RNAi When Electroporated into Several Primary Cell Types. Three siRNAs (1.33 µg) targeting CDK2, p53, JAK1 and a scrambled sequence were electroporated into HUVEC, NHEK and NHDF-neo human primary cells. 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 expression was calculated as a percentage of gene expression compared with the negative control siRNA. Electroporations were performed in duplicates.

Conclusions

A newly developed transfection agent provides a convenient and effective means to transfect normal cell lines with very low amounts of siRNA with minimal cytotoxicity.
Electroporation provides an efficient non-viral method for delivering siRNAs to primary and hard-to-transfect cells.
High cell viability and successful gene silencing in primary cells can be achieved by systematically addressing several electroporation parameters.