1. GFP (%) GenJet Fugene L2K Amaxa Figure1. A comparison study showing exceptional efficiency of GenJet™ reagent on HepG2 cells. Application Note Explorer, 2011, Vol 1, Issue 3 Improved Transfection of HepG2 Cells Using GenJet™ Transfection Reagent Introduction HepG2 is a perpetual cell line derived from the liver tissue of a 15 year old Caucasian American male with a well differentiated hepatocellular carcinoma [1]. Because of their high degree of morphological and functional differentiation in vitro, HepG2 cells are a suitable model to study the intracellular trafficking and dynamics of bile canalicular and sinusoidal membrane proteins and lipids in human hepatocytes in vitro [2]. HepG2 cells are also frequently used for studying expression and regulation of liver-specific genes [2]. However, one problem associated with using HepG2 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 24% and 15% efficiency respectively. In the present study, we optimized transfection conditions for GenJet DNA transfection reagent version II (Catalog # SL100489) and achieved average 76% efficiency in HepG2 cells as judged by sorting and counting GFP+ cells. Results Transient transfection of GFP plasmid DNA (pEGFP-N3, 2 µg per well) was performed on 6-well plate at optimal 70% confluency of HepG2 cells. In comparison of GenJet™ reagent (Ver. II), transient transfections were also performed in parallel with leading transfection products such as lipofetcamine 2000 (L2K), Fugene 6 and Amaxa electroporation per manufacturers’ protocols. GFP expression in HepG2 cells was measured by flow cytometry 36 hours post transfection. The present comparison study showed that GenJet™ gave average 76% GFP+ HepG2 cells (Figure 1) without sacrificing cell viability, outperforming Fugene 6 (average 15% efficiency) and L2K (average 24% efficiency). Amaxa was found to deliver excellent efficiency with average of 73% GFP+ HepG2 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 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 hepG2 cells. In contrast to the liposome reagents, GenJet™ Ver. II, formulated with a novel chemistry, gave exceptional transfection efficiency in HepG2 cells. In consistency with our finding, Dr. Uhart utilized GenJet™ Ver. II to successfully deliver a 30 Kb plasmid DNA to HepG2 cells with satisfied efficiency [3]. In addition, GenJet™ Ver. II was confirmed to be able to deliver shRNA to HepG2 cells with more than 80% gene silencing [4]. Though Amaxa gave comparable efficiency on HepG2 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 HepG2 cells. References 1. Knowles et al. Science, 1980; 209: 497 2. Sven et al. J Cell Biol., 1998; 142: 683–696 3. Uhart et al. J. Mol Bio., 2011; 406: 552-557 4. Li et al. Neoplasia, 2010; 12: 150–160 ProductCatalog #SizePrice GenJet™ Ver. IISL1004895 x 1.0 ml$741 1.0 ml$156 0.5 ml$86 Ordering Info