1. GFP (%) GenJet Fugene L2K Amaxa Figure1. A comparison study showing exceptional efficiency of GenJet™ reagent on C2C12 cells. Application Note Explorer, 2011, Vol 1, Issue 4 Efficient Transfection of C2C12 Cells Using GenJet™ Transfection Reagent Introduction C2C12 is a mouse myoblast cell line. C2C12 cells were originally obtained by Yaffe and Saxel through serial passage of myoblasts cultured from the thigh muscle of C3H mice [1]. These cells are capable of differentiation. C2C12 cells are a useful tool to study the differentiation of myoblast and osteoblast, to express various proteins, and to explore mechanistic pathways [1]. However, one problem associated with using C2C12 cells as an in vitro transient expression assay system is that high transfection efficiency is difficult to achieve with many commonly used chemical methods. Among the methods we have extensively tested, two leading products such as lipofectamine 2000 and Fugene 6 gave only ~3% and ~1% efficiency respectively. In the present study, we optimized transfection conditions for GenJet DNA transfection reagent version II (Catalog # SL100489-C2C12) and achieved average 65% efficiency in C2C12 cells as judged by sorting and counting GFP+ C2C12 cells. Results A so-called “shaved cell transfection” protocol was employed to transiently transfect C2C12 cell by delivering a GFP plasmid DNA (pEGFP-N3, 2 µg per well) with GenJet™ reagent. Briefly C2C12 at 95~100% confluency was suspended by trypsinization followed by centrifuge to prepare cell pellet, which then incubated immediately with transfection complex of GenJet™ reagent and pEGFP- N3 at 37 0 C. Transient transfections were also performed in parallel with leading transfection products such as lipofetcamine 2000 (L2K), Fugene 6 and Amaxa electro- poration per manufacturers’ protocols. GFP expression in C2C12 cells was measured by flow cytometry 36 hours post transfection. The present comparison study showed that GenJet™ gave average 65% GFP+ C2C12 cells (Figure 1) without significantly sacrificing cell viability, outperforming Fugene 6 (average 2% efficiency) and L2K (average 5% efficiency). Amaxa was found to deliver excellent efficiency with average of 68% GFP+ C2C12 cells. However large number of cell death was observed 36 hours post electroporation with Amaxa. www.signagen.comwww.signagen.com Toll-free: 866.918.6812 Ordering Info Discussion GenJet™ transfection reagent was developed by cross- linking a lipid to the backbone of a polymer. With an unique chemistry, GenJet™ Version II was further formulated by releasing all the DNA condensing groups, giving rise to 3~20 times better efficiency than its previous version. Per our validation data, GenJet™ Ver. II shows significant different mammalian cell transfection spectrum compared with liposome or lipid based transfection reagents like Fugene 6 and L2K. We have tried most of liposome reagents in the market and failed to succeed on C2C12 cells. In contrast to the liposome reagents, GenJet™ Ver. II, formulated with a novel chemistry, gave exceptional transfection efficiency in C2C12 cells with a pre-optimized protocol. In consistency with our finding, Dr. Kazgan utilized GenJet™ Ver. II to successfully deliver a 30 Kb plasmid DNA to C2C12 cells with satisfied efficiency [2]. Though Amaxa gave comparable efficiency on C2C12 cells in the present comparison study, heavy cell death caused by electroporation will eventually affect data acquisition and results interpretation. In conclusion, GenJet™ Ver. II provides a proven and satisfied method to deliver plasmid DNA to C2C12 cells. References 1. Yaffe D, et al. Nature, 1977; 270: 725–727 2. Kazgan K, et al. Mol. Biol. Cell, 2010; 21: 3433 - 3442 ProductCat #SizePrice GenJet™ Ver. II for C2C12 Cell SL100489-C2C125x1.0 ml$741 1.0 ml$156 0.5 ml$86