The Efficiency of CD40 Down Regulation by siRNA and Antisense ODN: Comparison of Lipofectamine and FuGENE6.
Dendritic cells (DCs) are ideal accessory cells in the field of gene therapy. Delivery of DNA and siRNA into mammalian cells is a useful technique in treating various diseases caused by single gene defects. Selective gene silencing by small interfering RNAs (siRNAs) and antisense oligodeoxynucleotides (ODN)s is an efficient method for the manipulation of cellular functions. An efficient, functional delivery system with no toxicity problems would be attractive.



We compared two commercially available cationic lipids, Lipofectamine and FuGENE6, in the delivery of both siRNA and antisense ODNs into mice spleen-derived DCs.



Cellular uptake was measured by the means of fluorescein-labelled non-silencing siRNA and antisense ODNs as a model system using flow cytometry. Cytotoxicity of the two delivery systems was compared with propidium iodide and annexin-V staining, and quantified with flow cytometry. The efficiency of our oligonucleotide delivery systems was compared by measuring CD40 expression by flow cytometry.



CD40 expression in DCs was 38%. After siRNA transfection by Lipofectamine, CD40 expression decreased to 13%, and after transfection by FuGENE6, it decreased to 18%. The difference was statistically significant. CD40 down regulation in DCs transfected with the two different antisense sequences by Lipofectamine was 21% and 23%, and down regulation after transfection by FuGENE6 was 19% and 18%, respectively. The differences were not statistically significant. The effects of siRNA and antisense ODNs were specific.



Lipofectamine was a more potent delivery system in siRNA effect, followed by FuGENE6. There was no significant difference between Lipofectamine and FuGENE6 as a delivery system of antisense ODNs.
Ebadi
Padideh
P
Department of Biology, Science and Research Branch, Islamic Azad University.
Karimi
Mohammad Hossein
MH
Pourfathollah
Ali Akbar
AA
Saheb Ghadam Lotfi
Abbas
A
Soheili
Zahra Soheila
ZS
Samiee
Shahram
S
Hajati
Smerdis
S
Nadali
Fatemeh
F
Geramizadeh
Bita
B
Moazzeni
Seyyed Mohammad
SM
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D019013:CD40 Antigens / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D003713:Dendritic Cells / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics*; D007202:Indicators and Reagents / Q000737:chemistry; D008055:Lipids / Q000737:chemistry*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013154:Spleen / Q000166:cytology; D014162:Transfection / Q000379:methods*
D019013:CD40 Antigens; C411955:FuGene; D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D020319:Oligodeoxyribonucleotides, Antisense; D034741:RNA, Small Interfering