Lipid-mediated gene delivery to HSC-3 and HEK293 cells in the presence of serum
Nejat Düzgüneş: 0000-0001-6159-1391
31st Annual Meeting of the American Association for Dental Research
San Diego, CA
March 6-9, 2002
Date of Presentation
Journal of Dental Research
Journal Volume Number
Oral squamous cell carcinoma (OSCC) is the most common tumor involving the oral cavity and oropharynx. Our long-term goal is to deliver the Herpes Simplex Virus thymidine kinase (HSV-tk) "suicide gene" for the treatment of OSCC. Cationic lipid-DNA complexes (lipoplexes) are being used increasingly as reagents for gene delivery both in vivo and in vitro. One limitation of the use of lipoplexes in vivo is the inhibition of gene delivery by serum. We therefore examined whether lipoplexes formed with transferrin (Tf)-complexed DOTAP/DOPE (Escort) or with Fugene could transfect human squamous cell carcinoma (HSC-3) and human embryonal kidney (HEK293) cells in the presence of high concentrations of fetal bovine serum (FBS). The pCMVluc plasmid encoding luciferase was complexed with these reagents, and incubated with the cells for 4 h at 37ºC in media containing various FBS concentrations. The medium was then replaced and cells were incubated for 2 days in medium with 10% FBS, after which luciferase activity was determined in a luminometer. Cell viability, quantified by the Alamar Blue assay, was higher than 90% in all transfected wells. In the presence of serum, gene expression mediated by Escort+Tf was much more efficient than that by plain lipoplexes. The effect on serum of lipofection was dose-dependent. In the presence of 20% FBS, luciferase expression was 25-60% of controls without serum for Escort+Tf, and ~70% for Eugene. In the presence of 60% FBS, gene expression was 10-50% of controls for Escort+Tf, and 40-70% for Fugene. Thus, (i) complexation of Escort with Tf overcomes the inhibitory effect of serum on transfection and (ii) the use of Escort+Tf or Fugene facilitates transgene expression even in the presence of high concentrations of serum.
Lipid-mediated gene delivery to HSC-3 and HEK293 cells in the presence of serum.
Paper presented at 31st Annual Meeting of the American Association for Dental Research in San Diego, CA.