Efficient removal of LoxP-flanked genes by electroporation of Cre-recombinase mRNA.
Introduction of Cre-recombinase in target cells is currently achieved by transfection of plasmid DNA or by viral-mediated transduction. However, efficiency of non-viral DNA transfection is often low in many cell types, and the use of viral vectors for transduction implies a more complex and laborious manipulation associated with safety issues. We have developed a non-viral non-DNA technique for rapid and highly efficient excision of LoxP-flanked DNA sequences based on electroporation of in vitro transcribed mRNA encoding Cre-recombinase. A K562-DSRed[EGFP] cell line was developed in order to measure Cre-mediated recombination by flow cytometric analysis. These cells have a stable integrated DSRed reporter gene flanked by two LoxP sites, and an EGFP reporter gene, which could only be transcribed when the coding sequence for DSRed was removed. The presented data show recombination efficiencies, as measured by appearance of EGFP-fluorescence, of up to 85% in Cre-recombinase mRNA-electroporated K562-DSRed[EGFP] cells. In conclusion, mRNA electroporation of Cre-recombinase is a powerful, safe, and clinically applicable alternative to current technologies used for excision of stably integrated LoxP-flanked DNA sequences.
Van den Plas
Dave
D
Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium.
Ponsaerts
Peter
P
Van Tendeloo
Viggo
V
Van Bockstaele
Dirk R
DR
Berneman
Zwi N
ZN
Merregaert
Joseph
J
D018274:Electroporation; D005434:Flow Cytometry; D017353:Gene Deletion; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D020014:K562 Cells; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D008164:Luminescent Proteins; D012333:RNA, Messenger; D014764:Viral Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases