514. mRNA Transfection Results in Efficient Overexpression of Transgenes in Leukemic Cell Lines and Hematopoietic Stem Cells

Abstract

DNA-Vector transfection of eukaryotic cells needs transport of the DNA to the nucleus for efficient transcription and subsequent translation. In suspension cells most DNA-transfection methods are either inefficient (e.g. lipofection) or stressful for the cells (electroporation or nucleofection), which leads to high cell losses during the transfection process. We report the use of in vitro transcribed, capped and polyadenylated mRNA for the transfection of leukemic cell lines in comparison to DNA delivery. Templates for in vitro transcription were generated by PCR. The forward primer included the T7-promoter and the reverse primer a fragment of the beta-globin 3'UTR to increase the in vivo half life of the mRNA. Electroporation of GFP-mRNA into Jurkat, K562 or Kg1a lead to a GFP overexpression in 95 % of the transfected cells, with survival rates of 80 to 90%. Electroporation with pEGFP-N1 using the same pulse conditions resulted in GFP-overexpression in 62% of Jurkat or 9% of K562, with survival rates of 50% and 40 %, respectively. Our results suggest that electroporation of DNA is more toxic than mRNA transfection. GFP-expression in mRNA transfected cells was visible two hours after the transfection and peaked at about 12h. In contrast to DNA-transfection, mRNA mediated GFP expression levels were uniform in transfected cells. To investigate whether mRNA transfection results in functional transgene expression, mRNA coding the chemokine receptor CXCR4 was generated and electroporated into CXCR4 negative K562 cells. Flow cytometric analyses showed CXCR4 overexpression in more than 90% of transfected cells. Calcium flux measurements displayed a robust increase of intracellular free calcium in CXCR4-mRNA transfected K562 upon binding of the CXCR4 ligand SDF-1, while no calcium flux was visible in GFP-mRNA transfected or untransfected K562. Electroporation of GFP-mRNA into CD34+ hematopoietic progenitor cells or mesenchymal stem cells resulted in GFP overexpression in > 50% of transfected cells, respectively. Experiments are under way to further improve the mRNA transfection for primary cells. mRNA transfection is a highly efficient means for transient overexpression of transgenes in various cell lines and primary cells.