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Serum decreases the size of Metafectene-and Genejammer-DNA complexes but does not affect significantly their transfection activity in SCCVII murine squamous cell carcinoma cells

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Abstract

Cationic liposome-DNA (lipoplexes) or polymer-DNA (polyplexes) complexes have been used to deliver therapeutic genes, both in vitro and in vivo. However, gene transfer by these non-viral vectors is usually inhibited by biological milieu. A relatively high efficiency of transfection could be achieved in human oral cancer cells transfected with the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer, in the presence of 60% fetal bovine serum (FBS) (Konopka et al., Cell. Mol. Biol. Lett. 10 (2005) 455–470). Here, we examined the efficacy of these vectors to deliver β-galactosidase (β-gal), luciferase and Herpes Simplex Virus thymidine kinase (HSV-tk) genes to SCCVII murine squamous cell carcinoma cells, which are used to generate an orthotopic murine model of oral cancer. We also evaluated the hydrodynamic size and zeta potential of the vectors and the effect of FBS and mouse serum (up to 60%) on the size of Metafectene and GeneJammer complexes with the pCMV.Luc plasmid. Our results indicate that Metafectene and GeneJammer are highly effective in transfecting SCCVII cells. Approximately 60–70% of SCCVII cells transfected with pCMV.lacZ were positive for β-gal staining. The expression of β-galactosidase was essentially not affected by serum. Mouse serum (20–60%) reduced both Metafectene-and GeneJammer-mediated luciferase expression by 30–45%, while FBS did not affect transfection efficiency. The delivery of the HSV-tk gene by Metafectene or GeneJammer in the presence of 0% or 60% FBS, followed by GCV treatment for 6 days, resulted in over 90% cytotoxicity. The mean diameters of the DNA complexes of Metafectene and GeneJammer decreased significantly as a function of the serum concentration. The reduction in the size of the lipoplexes and polyplexes by serum was essentially not inhibitory to transfection of SCCVII cells. This is in contrast to previous hypotheses that serum-induced decrease in the size of lipoplexes is the primary cause of serum inhibition of transfection.

Abbreviations

β-Gal:

β-galactosidase

DMEM:

Dulbecco’s modified Eagle’s MEM medium

FBS:

fetal bovine serum

GCV:

ganciclovir

HBS:

HEPES-buffered saline

HNSCC:

head and neck squamous cell carcinoma

HSV-tk :

Herpes Simplex Virus thymidine kinase gene

OSCC:

oral squamous cell carcinoma

RLU:

relative light units

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Correspondence to Krystyna Konopka.

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Key words

  • Transfection
  • Metafectene
  • GeneJammer
  • Serum inhibition
  • HSV-tk gene
  • SCCVII murine squamous cell carcinoma cells