Novel non-viral method for transfection of primary leukemia cells and cell lines

Abstract

BACKGROUND: Tumor cells such as leukemia and lymphoma cells are possible targets for gene therapy. However, previously leukemia and lymphoma cells have been demonstrated to be resistant to most of non-viral gene transfer methods. METHODS: The aim of this study was to analyze various methods for transfection of primary leukemia cells and leukemia cell lines and to improve the efficiency of gene delivery. Here, we evaluated a novel electroporation based technique called nucleofection. This novel technique uses a combination of special electrical parameters and specific solutions to deliver the DNA directly to the cell nucleus under mild conditions. RESULTS: Using this technique for gene transfer up to 75% of primary cells derived from three acute myeloid leukemia (AML) patients and K562 cells were transfected with the green flourescent protein (GFP) reporter gene with low cytotoxicity. In addition, 49(+/- 9.7%) of HL60 leukemia cells showed expression of GFP. CONCLUSION: The non-viral transfection method described here may have an impact on the use of primary leukemia cells and leukemia cell lines in cancer gene therapy.

Figure 1

Primary data of GFP and eGFP transfected leukemic cell lines. Lymphocytes were gated based on their scatter profile (figures on the right) and cells were evaluated for transgene expression. a) GFP-expression of K562 cells. Cells were transfected with Ad-GFP at an MOI of 200 and assayed by flow cytometry 72 hours posttransfection. The overlay of the shaded histogram represents the background fluorescence of untreated cells. Positive transfected cells expressed intracellular green fluorescent protein. b) eGFP-expression of K562 cells 24 hours after nucleofection. c) eGFP-expression of HL60 cells 24 hours after nucleofection. Data are shown from one representative experiment.

Figure 2

Nucleofection mediated gene transfer in primary leukemic cells. eGFP expression in AML cells after exposure to optimized pulses. After 24 hours cells were harvested and analyzed by flow cytometric analysis. a) Representative flow cytomeric analysis for each of the three patients. Control cells were pulsed without DNA and showed no eGFP expression (left side); percentage of positive transfected cells is shown on the right. b) Gated and ungated transfection efficiencies of the primary AML cells. Percentage of dead cells was determined by PI staining. The figure represents data from two experiments, respectively. Data are presented as mean +/- standard error of the mean.

Figure 3

Nucleofection mediated gene transfer in leukemia cell lines. eGFP expression in K562 and HL60 cells after exposure to the optimized pulses. After 24 hours cells were harvested and analyzed by flow cytometric analysis. GFP positivity was assayed in gated as well as in ungated cell populations. Control cells were pulsed without DNA and showed no eGFP expression. Percentage of dead cells was determined by PI staining. The figure represents data from five separate experiments, respectively. Data are presented as mean +/- standard error of the mean.

Figure 4

Growth curves of K562 (a) and HL60 (b) cells, transfected by nucleofection technique. Cell proliferation was measured by trypan blue staining and cell count. The figure represents data from five separate experiments. Data are presented as mean +/- standard error of the mean.