HepG2, luciferase

Polyfection was carried out with pCMVLuc (a 5 kb pDNA) on HepG2 cells for four hours at 37 °C. After 48 hours, the transfection efficiency was determined from Luciferase activity in cells measured by luminescence (RLU) and the cytotoxicity was evaluated by using the colorimetric MTT assay, dp is the poly lysine degree of polymerization. The transfection efficiency is scored on a 0 to 100 scale where 0 and 1 00 correspond to an absence of transfection and the most effective transfection, respectively compared to the pDNA alone. His is histidyl residue. 

Figure 16.6 Polyfection mediated by a disulfide-containing poly lysine conjugate. (A) 293-T7 cells and (B) HepG2 cells were incubated for four hours at 37 °C with pCMVluc (5 pig) complexed with either disulfide-containing polylysine (20 pig) or polylysine (15 pig) in the presence of 10% FCS and 100 iM chloroquine. The luciferase activity was measured after 48 hours culture and expressed as the relative light units (RLU) per 106 cells.

Figure 4 Luciferase expression 48 h after transfection of HepG2 cells with an AVE/ pLuc complex in 10% serum as a function of DNA concentration. Experimental conditions were as described in Figure 3. Luciferase transgene expression was analyzed with a Pro-mega luciferase assay system, and samples were read in a Tropix TR717 microplate lumi-nometer. Cellular protein was measured using a Bio-Rad protein assay kit.

Figure 5 Luciferase expression 48 h after transfection of HepG2 cells with an AVE/ pLuc complex in 10% serum as a function of time. Experimental conditions were as in Figure 4. 

Westerink WM, Stevenson JC, Horbach GJ, Schoonen WG (2010) The development of RAD51C, Cystatin A, p53 and Nrf2 luciferase-reporter assays in metabolically competent HepG2 cells for the assessment of mechanism-based genotoxicity and of oxidative stress in the early research phase of drug development. Mutat Res 696 21 0... 

Figure 12 In vitro evaluation of the lipidoid library for sIRNA delivery, (a) Percent reduction of firefly luciferase expression in HeLa cells with lipidoid/ siRNA formulations. The data are split into five groupings spanning 0-100% luciferase reduction for ease of analysis, (b) Optimal knockdown levels in HeLa determined by screening at four different lipidoid/siRNA ratios, (c-e) Dose response silencing in HeLa (c), HepG2 (d), and primary macrophage (e) cells. Reprinted from Akinc, A. Zumbuehl, A. Goldberg, M. et at. Nat. Biotechnol. 2008, 26 (5), 561-569, with permission from the Nature Publishing Group.

In transfection experiments, lipid L2 and DOTAP were used to form lipoplexes and their ability to transfect HepG2 cells were assessed. The lipoplexes were formed around expression plasmids encoding firefly luciferase and a strong promoter to monitor transfection efficiency. Lipoplexes were incubated with cells for 4 h and luciferase luminescence was quantitated. DNA/L2 complexes showed low luciferase expression however, mixtures of L2 and DOPE complexed with DNA showed improvement and matched that of DOTAP. Further optimization of the lipid/DNA ratio yielded complexes with similar transfection efficiency of other cationic lipid delivery systems. To address the problem of serum proteins degrading lipoplexes, the experiment was repeated for L2 with cells complimented with 25% fetal calf serum. After 24 h, luciferase expression was monitored and it was found that all fiuorinated lipoplexes retained or increased their transfection efficiency, compared to DOTAP whose performance saw a 30-40% reduction. 

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