Nucleofection as an efficient nonviral transfection method for human monocytic cells

Martinet W, Schrijvers DM and Kockx MM
Source: Biotechnol Lett
Publication Date: (2003)
Issue: 25: 1025-1029
Cells used in publication:
Species: human
Tissue Origin: blood
Species: human
Tissue Origin: blood
The authors evaluated the Nucleofector technology as an efficient non-viral transfection method for human monocytic cells compared to standard electroporation. The commonly used monocytic cell lines U937 and THP-1 were nucleofected with either an eGFP expression plasmid or eGFP encoding mRNA. The authors demonstrated that nucleofection is superior to standard electroporation. Nucleofection yielded up to 80% transfection efficiency. Compared to standard electroporation efficiency for DNA transfection was improved by ~50% and 30% for U937 and THP-1, respectively. Also the transfer efficiency of eGFP-specific mRNA after nucleofection increased with approximately 20% compared to standard electroporation. Western blotting of protein lysates from eGFP transfected cells showed detectable eGFP expression as early as 2 hours after nucleofection. The initiation of cell death was low (2.5-fold increase of propidium iodide incorporation of DNA nucleofected cells versus non-transfected cells), or even absent (in case of mRNA nucleofection).
Despite some progress in the field of gene transfer into hard-to-transfect cells, so far an efficient nonviral method for monocytes has not been available. A comparison of plasmid DNA with capped and polyadenylated mRNA for enhanced green fluorescent protein gene delivery into the commonly used monocytic cell lines U937 and THP-1 suggested that limited DNA trafficking may be the underlying cause of poor transfection results. As Nucleofector technology delivers DNA (or mRNA) straight into the nucleus, we obtained nucleofection efficiencies of up to 80% without significant cell toxicity. Moreover, as the DNA quickly reaches the nucleus, nucleofected cells were ready for analysis after only 2-6 h. The technique is suitable not only for monocytes but also for other hard-to-transfect cells.