Abstract
mRNA transfection is a safe alternative to DNA transfection for transient transgene overexpression in mammalian cells as genomic integration of the transferred nucleic acid is impossible. We developed a new mRNA electroporation protocol for transfection of primary mesenchymal stem cells (MSCs) using a hypoosmolar buffer system. Polyadenylated and capped mRNA was in vitro transcribed from DNA templates by T7 RNA polymerase. The DNA template generation has been simplified by our group using PCR amplification of the transgene sequence with primers providing the T7 RNA polymerase promoter and optimized 5′ and 3′ UTR sequences for better translation and RNA stability. This manoever allows to generate the DNA template from any plasmid vector and bypasses the need for additonal cloning steps. Canine primary MSCs were isolated by selection of adherent cells from bone marrow aspirates in a low glucose medium. Transfection of GFP or CXCR4 mRNA into primary MSCs resulted in transgene overexpression in more than 90% of target cells with excellent viabilities (>95%). Expression of CXCR4 was well detectable for up to 96 hours after transfection. SDF-1 dependent Ca-flux was only present in CXCR4-transgene overexpressing MSCs suggesting functional expression of the chemokine receptor. Transwell migration experiments demonstrated a 2 fold increased migration of CXCR4 mRNA transfected MSCs towards a gradient of SDF-1 compared to background migration without SDF-1. GFP mRNA transfection did not result in any SDF-1 stimulated migration. Our data demonstrate efficient and safe transient genetic engineering of primary MSCs based on mRNA transfection. mRNA transfection induced chemotaxis will facilitate receptor mediated targeting of transplanted MSCs at specific body compartments.
Mono Q chromatography permits recycling of DNA template and purification of RNA transcripts after T7 RNA polymerase reaction
