Efficient generation of stably electrotransfected human hematopoietic cell lines without drug selection by consecutive FACsorting.

Background: Current methods to establish stably transfected cell lines by nonviral techniques involve coselection for a drug selection marker. However, this approach suffers from several drawbacks. We developed a fluorescence-activated cell sorting (FACS)-based protocol for the selection and isolation of stable hematopoietic electrotransfectants without the need for selective growth conditions.

Methods: Leukemic K562 cells were electroporated with the enhanced green fluorescent protein (EGFP) reporter gene and FACsorted to obtain stably EGFP-expressing cells. Stable EGFP(+) clones were established by single-cell sorting.

Results: Efficiency of stable EGFP gene expression increased steadily in function of number of consecutive FACsorts. Stable transfectants (>99% EGFP(+)) were obtained after four FACsorts. Furthermore, several single-cell derived clones with variable levels of stable EGFP expression were isolated and cultured without the use of selective growth media.

Conclusions: EGFP is an effective selection marker for the generation and isolation of stably transfected hematopoietic cell clones without the need for selection in toxic media that could create a potentially undesirable stress environment for stably transfected cells.