Plasmid DNA and siRNA transfection of intestinal epithelial monolayers by electroporation.
This study was conducted to evaluate the ability of electroporation to efficiently transfect differentiated intestinal epithelial monolayers with plasmid DNA and to determine whether electroporation can transfect these monolayers with short-interfering RNA (siRNA) to cause gene silencing. Confluent T84 monolayers were transfected with reporter plasmids expressing luciferase or green-fluorescent protein or with siRNA directed against the nuclear envelope proteins lamin A/C using electroporation. Optimized electroporation conditions resulted in luciferase and GFP expression. Both intracellular uptake of fluorescently labeled plasmid and expression of the reporter genes increased with increasing electroporation strength and DNA concentration. When monolayers were transfected by lipofection with the reporter plasmids, expression and DNA uptake were less than for electroporation. Electroporation was also found to transfect monolayers with siRNA, which resulted in up to 90% inhibition of targeted protein production. Silencing occurred within 24h of transfection and increased with increasing siRNA concentration. These results suggest that electroporation can provide a valuable research tool for transfection of intestinal epithelial monolayers and other differentiated cell systems, and may ultimately be useful for clinical gene therapy applications.
The Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Georgia Institute of Technology, Atlanta, GA 30332, USA.
Esi B
EB
Ghartey-Tagoe
Brian A
BA
Babbin
Asma
A
Nusrat
Andrew S
AS
Neish
Mark R
MR
Prausnitz
D002460:Cell Line; D018274:Electroporation / Q000379:methods* / Q000592:standards; D004847:Epithelial Cells / Q000378:metabolism; D020868:Gene Silencing; D017930:Genes, Reporter; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology*; D010957:Plasmids / Q000235:genetics*; D011506:Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection / Q000379:methods* / Q000592:standards
D011506:Proteins; D034741:RNA, Small Interfering