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.
Van Tendeloo
V F
VF
Laboratory of Experimental Hematology, University of Antwerp (UIA), Antwerp University Hospital (UZA), Edegem, Belgium.
Ponsaerts
P
P
Van Broeckhoven
C
C
Berneman
Z N
ZN
Van Bockstaele
D R
DR
D000900:Anti-Bacterial Agents; D003001:Cloning, Molecular / Q000379:methods; D018274:Electroporation; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression / Q000276:immunology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D019169:Jurkat Cells / Q000166:cytology*; D020014:K562 Cells / Q000166:cytology*; D008164:Luminescent Proteins / Q000235:genetics; D010957:Plasmids; D014162:Transfection / Q000379:methods*; D020298:U937 Cells
D000900:Anti-Bacterial Agents; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins