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Relationship between plasmid DNA topological forms and in vitro transfection

P Chancham1, J A Hughes

  • 1Department of Pharmaceutics, University of Florida College of Pharmacy, P.O. Box 100494, Gainesville, FL, 32603, U.S.A.

Journal of Liposome Research
|June 18, 2009
PubMed

Insights

The open circular form of plasmid DNA shows higher transfection efficiency and cellular association compared to supercoiled or linear forms. This DNA topoisomer stability varies in cell lysate, with supercoiled DNA being least stable.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Plasmid DNA (pDNA) exists in various topological forms, including supercoiled, open circular, and linear.
  • These topological isomers may influence pDNA's biological activity, particularly transfection efficiency.
  • Understanding these differences is crucial for optimizing gene delivery systems.

Purpose of the Study:

  • To investigate the relationship between major pDNA topoisomers (supercoiled, open circular, linear) and their biological activity.
  • To compare the transfection efficiency of different pDNA topoisomers using cationic liposomes (DOTAP/DOPE) and electroporation.
  • To assess the stability of pDNA topoisomers in cell lysate and their cellular association.

Main Methods:

  • Transfection of Chinese Hamster Ovary (CHO) and SKnSH cells with pDNA topoisomers using DOTAP/DOPE liposomes or electroporation.
  • Incubation of pDNA topoisomers in NP40 cell lysate at 37°C to determine stability.
  • Flow cytometry analysis of fluorescein-labeled pDNA topoisomer association with CHO cells after liposome-mediated delivery.

Main Results:

  • The open circular form of pCMV-luciferase exhibited higher transfection efficiency than supercoiled or linear forms.
  • Plasmid pGL3 showed no significant difference in transfection efficiency across topoisomers.
  • Supercoiled pDNA was least stable in cell lysate (detected up to 4 min), while open circular and linear forms had half-lives of 6 and 10 min, respectively.
  • Open circular pDNA demonstrated greater cellular association than linear forms when delivered via cationic liposomes.

Conclusions:

  • All three studied pDNA topoisomers are transcriptionally active.
  • Transfection efficiency and cellular association patterns vary among pDNA topoisomers, with the open circular form often showing advantages.
  • The stability of pDNA topoisomers in cellular environments differs, impacting their biological availability.

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