Flow-cytometric analysis of mouse embryonic stem cell lipofection using small and large DNA constructs.
Using the lipofection reagent LipofectAMINE 2000 we have examined the delivery of plasmid DNA (5-200 kb) to mouse embryonic stem (mES) cells by flow cytometry. To follow the physical uptake of lipoplexes we labeled DNA molecules with the fluorescent dye TOTO-1. In parallel, expression of an EGFP reporter cassette in constructs of different sizes was used as a measure of nuclear delivery. The cellular uptake of DNA lipoplexes is dependent on the uptake competence of mES cells, but it is largely independent of DNA size. In contrast, nuclear delivery was reduced with increasing plasmid size. In addition, linear DNA is transfected with lower efficiency than circular DNA. Inefficient cytoplasmic trafficking appears to be the main limitation in the nonviral delivery of large DNA constructs to the nucleus of mES cells. Overcoming this limitation should greatly facilitate functional studies with large genomic fragments in embryonic stem cells.
McLenachan
Samuel
S
Murdoch Childrens Research Institute, Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia.
Sarsero
Joseph P
JP
Ioannou
Panos A
PA
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004274:DNA, Recombinant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D007202:Indicators and Reagents; D008055:Lipids; D008081:Liposomes; D051379:Mice; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D013844:Thiazoles; D014162:Transfection / Q000379:methods*
D004274:DNA, Recombinant; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; C086329:TOTO-1 thiazole orange dye; D013844:Thiazoles; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins