, 2003 and Schnoor et al., 2009). Recently, others have demonstrated the successful transfection of mRNA into primary murine and human monocytes using mouse macrophage Nucleofector and Human Monocyte Nucleofector kits, INK128 respectively (Zimmermann et al., 2012). Although our methods (specifically
the kit used) are similar to the published study, our unsuccessful attempts could illustrate the need for a cell-type specific Nucleofector kit, optimized for primary rat monocytes, in order to achieve effective transfection. It could also be possible that mRNA transfection is more potent for primary monocyte nucleofections. Despite exhaustive optimization attempts, classical transfection methods (i.e. lipid-based reagents, electroporation,
and nucleofection) were unable to generate stable NGF expression in primary rat monocytes. Although nucleofection generated some NGF expression in monocytes (0.8 ± 0.2 ng/ml NGF per 24 h per 1 million cells), reproducibility was highly variable (21% successful). Even after exhaustive attempts at optimizing the transfection conditions (i.e. plasmid purity, cell purity, various incubation and culture conditions, etc.), we were unable to achieve Selleck LDK378 better reproducible results. Since our interest is to later administer NGF-secreting monocytes in vivo, we concluded that this method would not serve as an attractive method for future experiments. In this study, we demonstrated that lentiviral vectors and Bioporter were the most efficient methods for generating NGF-secreting monocytes. Others have also reported success using viral transduction methods in these cells. Herold et al. (2006) demonstrated that adenoviral infection transduced approximately 95%
of primary monocytes and Mordelet et al. (2002) demonstrated the success and efficiency of lentiviral transduction mafosfamide for monocyte/macrophage gene delivery in rats. In this study, monocytes transduced with lentiviral vector pHR-ba-NGF or pHR-SFFV-NGF produced 15.6 ± 2.5 or 9.1 ± 2.6 ng/ml per 1 million cells, respectively. Both exhibited high reproducibility at 100% and 86%, respectively. Thus, our data is in line with others supporting the use of viral transduction for successful DNA delivery to primary monocytes. Since the use of lentiviral vectors still remains controversial due their immunogenicity properties and we ultimately plan on using these cells for in vivo studies, we also investigated Bioporter as a nonviral approach for generating NGF-secreting monocytes. Bioporter is a protein delivery system that relies on lipid complexes to translocate proteins into target cells. Previous investigations have established that this system can effectively deliver functional recombinant proteins to a wide variety of cell types (Böttger et al., 2010). In the present study, we demonstrate that Bioporter is an efficient nonviral method to deliver NGF to primary rat monocytes, in which monocytes secrete 0.6 ± 0.