Applications

Accell siRNA

Kirsteen MacLean, Ph.D.

Like many bench researchers, I spent numerous frustrating months figuring out options to transfect siRNA into my primary and various hematopoeitic cells. Earlier this year Accell siRNA technology was released representing a novel technology that solves these delivery challenges by permitting the absorption of siRNA directly into cells without the need for lipid transfection reagents, viruses or electroporation. Thus, a breakthrough in siRNA delivery for difficult to transfect cell types was born. This latest silencing tool leverages innovations from earlier technologies developed by the scientists at Thermo Fisher Scientific. These technological advances are the foundation for ON-TARGETplus™ reagents for reduced off-target effects and siSTABLE® which enables siRNA knockdown in the presence of nuclease-rich environments.

While Accell siRNA was only released within the last year; several research groups have quickly embraced the advantages of Accell siRNA technology to address their scientific needs. Recent publications, three described herein citing the utility of Accell siRNA in traditionally difficult to transfect cells, bely the significance of this new technology for RNAi-based studies in clinically relevant biological models. This is particularly important for a population where the life expectancy in western countries has increased from around 50 to now 75 years during the last century with public health measures such as vaccinations and antibiotics that have greatly reduced deaths in childhood. However, with increased lifespan so have we seen an increase in associated developmental and age-related risk factors including neurodegeneration and cancer. Specifically, insight into the health and development of the brain and the reproductive tract remain ambiguous in large part because the relevant cell types are not easily manipulated in culture. Accell siRNA now offers researchers a novel tool with which to dissect critical molecular events in cell types associated with the healthy or affected tissues as evidenced by three reports reviewed here.

In a recent study published by Guy Breton’s group [1], Accell siRNA was used to investigate the role of G protein signaling interactions in the neuronal oligodendrocyte progenitor cell line, CG-4, which can be induced to differentiate into oligodendrocytes by replacing conditioned medium with differentiation medium. The G protein-coupled receptor class of thyrotropin receptors (TPR) are known to be involved in the development and function of the myelin-producing oligodendrocytes (OLGs) – deficiencies of which have profound effect on brain development and regenerative capacities. Studies using several different G proteins revealed for the first time that TPR’s were coupled to the Gs subunit in the nuclear compartment of mature OLG’s. The investigators used Accell siRNA in both progenitor CG-4 cells and mature OLG’s to specifically address the role of the Gs subunit and determined that knockdown of this subunit affected TPR-stimulated cAMP production and subsequent CREB phosphorylation, both of which had previously been shown to promote OLG survival. The investigators thus determined a novel, well-coordinated coupling of differential G protein expression and distribution to TPR’s during OLG development.

In other work published by the Nijholt lab [2], researchers employed Accell siRNA technology to help investigate the role of TNF-β activation and NF-κB signaling in neurodegeneration and to dissect the molecular mechanisms of TNF-β-mediated neuroprotection against glutamate-induced excitotoxicity in primary murine cortical neurons. The group focused their studies on the role of the NF-κB regulated calcium activated potassium channel (Kca) demonstrating that pre-treatment with Kca2.2 channel Accell siRNA reduced overall cell survival by significantly affecting TNF-β induced neuroprotection following challenge with glutamate. The results therefore bring a better understanding of neuronal survival during conditions of overstimulation.

Yet another recent report focused on ovarian cancer, currently the fifth leading cause of death from cancer in women. There are three main types of ovarian tumors: germ cell, stromal and the most common, epithelial which accounts for 85% to 90% of all ovarian cancer cases. Currently, no definitive biomarkers have been identified to improve early detection. Studies led by researchers at the University of Texas Southwest Medical Center [3] used various ovarian cancer cell lines and evaluated the role of Phospho-enriched protein in astrocytes (PEA-15), a 15-kDa phosphoprotein previously shown to slow cell proliferation by inhibiting ERK-dependent transcription and proliferation. Importantly, the group found that depletion of PEA-15 by Accell siRNA in high-PEA-15-expressing OVCA-432 ovarian cancer cells resulted in increased proliferation and colony formation. Conversely, expression of PEA-15 inhibited ovarian cell proliferation and promoted the induction of autophagy, the evolutionary conserved and ancient program of cell turnover. Notably, the clinical relevance of PEA-15 expression was revealed in patient biopsy tissue samples demonstrating correlation with improved overall survival. These results highlighted the importance of not simply targeting ERK-dependent pathways but also indicated the potential of PEA-15 as a novel biomarker in predicting patient outcomes.

Collectively these findings suggest the applicability of Accell siRNA to cell types traditionally resistant to other forms of transfection and pave the way to utilizing gene silencing in various cell types. By appreciating the molecular events important for cell development and cell health in both normal and aberrant cell types we can begin to address improved drug development and treatment options for many disease states.

References

1. Mir F, et al. A Novel Nuclear Signaling Pathway for Thromboxane A2 Receptors in Oligodendrocytes: Evidence for Signaling Compartmentalization during Differentiation. MCB, 2008. 28: 6329-6341. PubMed
2. Dolga AM, et al. TNF-α-mediates neuroprotection against glutamate-induced excitotoxicity via NF-κB-dependent up-regulation of Kca2.2 channels. J. Neurochemistry, 2008. 10: 1-10. PubMed
3. Bartholomeusz C, et al. PEA-15 induces autophagy in human ovarian cancer cells and is associated with prolonged overall survival. Cancer Res, 2008. 68: 9302-9310. PubMed