Optoinjection of siRNA and functional knockdown of erythropoietin receptor in primary human CD34+ cells

IntroductionDownload as PDF

RNA interference (RNAi) is a pathway for specific and efficient post-transcriptional gene silencing which is now in widespread use for a variety of applications. Despite the extensive utilization of RNAi through small interfering (si)RNA delivery, several limitations remain including: (i) inefficient introduction of siRNA into many important cell types; (ii) potential for off-target and toxic-response gene effects; and (iii) low cell viability and yield with standard transfection techniques.

To address current limitations with siRNA delivery, an alternative method based on laser-mediated, transient cell permeabilization (optoinjection) was assessed using the Laser-Enabled Analysis and Processing (LEAP™) system. Lasers have been used to deliver macromolecules into living cells for many years (Tsukakoshi et al. 1984, Tao et al. 1987; Guo et al. 1995; Shirahata et al. 2001; Tirlapur and Konig 2002), and LEAP has previously been shown to provide high-throughput optoinjection of a variety of macromolecules into living cells with high efficiency and viability (Clark et al. 2006). Importantly, optoinjection has proven to be relatively non-toxic compared to other transfection methods, particularly with cell types that are difficult to transfect via other means (e.g., B cells, T cells, neurons) (Clark et al. 2006).

The results shown here demonstrate that the LEAP Grid Optoinjection Application results in improved siRNA delivery into primary human CD34+ cells with low toxicity and high cell viability and yield, enabling functional genomics studies with these important cells. The ability to study gene function in primary human cells has the potential to provide more relevant information than can be obtained from cell line systems which do not accurately reflect human physiology in many cases.

Validation Approach

Hematopoietic stem cells (HSCs) are self-renewing, pluripotent, and can reconstitute all blood and immune cell populations. HSCs are found in bone marrow, peripheral blood, and umbilical cord blood and are studied extensively in a variety of applications. HSCs are generally difficult to transfect, and therefore primary human CD34+ cells provide a suitable model for testing the applicability of siRNA optoinjection. Optoinjection of siRNA directed against EpoR was used as a model to provide both mRNA- and functional-level knockdown readouts using standard qRT-PCR and burst forming unit-erythroid (BFU-E) differenitation assays (Pessina et al. 2005).

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  Fig. 1. Optoinjection of siRNA targeted to EpoR results in decreased mRNA expression. Primary human cord blood CD34+ cells were seeded into a 384-well plate and either optoinjected on LEAP with 350 nM siRNA or transfected with Lipofectamine™ 2000 according to manufacturer’s recommendations. Cells were processed 48 hours post-treatment for qRT-PCR analysis. Cells treated with EpoR siRNA were normalized to cells treated with a control non-specific siRNA. EpoR mRNA expression was also normalized to 18S RNA levels. Each data set represents eight replicates. The asterisk (*) indicates a p-value < 0.05 when compared to control cells treated with non-specific siRNA. Cell viability was excellent (>94%) after both treatments.

Results

The fully automated processing capability of LEAP was first used to screen multiple optoinjection parameters for siRNA delivery into primary human CD34+ cells in 384-well plates (not shown). The best condition was then used for subsequent experiments. Optoinjection of EpoR siRNA into primary human CD34+ cells using the LEAP Grid Optoinjection Application resulted in a 43.1% knockdown of EpoR mRNA levels (p = 0.001), as detected by qRT-PCR (Fig. 1). Notably, Lipofectamine™ 2000 transfection did not result in significant knockdown (p = 0.29). This experiment was repeated with cells from three different donor pools with similar results.

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  Fig. 2. Optoinjection of siRNA directed to EpoR results in decreased erythropoiesis in response to extracellular erythropoietin. Primary human cord blood CD34+ cells were seeded into a 384-well plate at 2000 cells per well. Cells were either optoin¬jected on LEAP with 350 nM siRNA or transfected with Lipofectamine™ 2000 ac¬cording to manufacturer’s recommendations. Cells were plated in methylcellulose containing medium and growth factors 48 hours post-treatment. Colonies were allowed to grow for 14 days and were scored as either red (erythrocyte) or white (non-erythrocyte) (see top panel). The ratio of red cells to white cells as a percent¬age of total cell population was compared between EpoR siRNA treated cells and cells treated with a control non-specific siRNA (bottom panel). Each data set rep¬resents eight replicates. The asterisk (*) indicates a p-value < 0.05 when compared to untreated control cells.

To determine if optoinjected siRNA was also able to affect protein function, cells were induced to differentiate toward the erythrocyte lineage. The number of BFU-E colonies that developed after siRNA optoinjection was reduced by 48% as compared to untreated controls (Fig. 2; p < 0.05). In contrast, optoinjection of non-specific siRNA did not alter BFU-E development (not shown). In addition, siRNA delivery by Lipofectamine™ 2000 did not affect BFU-E formation. In short, the functional assay results matched the qRT-PCR results, demonstrating that effective target silencing was obtained only by siRNA optoinjection in this important cell type.

Since the purpose of siRNA delivery is to study the effect of specific gene knockdown, off-target, and toxic gene effects as reported with lipid-mediated transfection could be of major concern. Cytotoxic effects could elicit a variety of stress responses in cells, changing gene expression patterns and affecting normal cell physiology. Therefore, the expression levels of five genes that cover a broad range of cell stress responses were examined in this study (Fig. 3). Optoinjection resulted in increased expression of Heat Shock Protein 70 mRNA, whereas Lipofectamine 2000 transfection demonstrated a much greater and diverse cytotoxicity.

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  Fig. 3. Optoinjection of stem cells does not trigger a significant change in expression of common stress-response genes when compared to transfection by Lipofectamine™ 2000. The same cell lysates for transfection of EpoR siRNA used for the experiment in Fig. 1 were also used to detect expression levels of the common stress response genes Clusterin, HSP70, MX1, GADD45, and MAPK1.

Conclusion

The LEAP system is a robust and relatively benign platform for optoinjection of functional siRNA into primary human CD34+ cells, resulting in decreased target mRNA (EpoR) and loss of associated gene function (BFU-E formation). The LEAP Grid Optoinjection Application can be performed in situ in a fully-automated, high-throughput manner using common cell culture plate formats. The low cytotoxicity effects of optoinjection may improve the biological relevance of data as compared to the more toxic methods currently in common use, particularly for challenging cell types.

References

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