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Nonhomologous end-joining ligation transfers DNA regulatory elements between cointroduced plasmids
Toshio Ishikawa1, Eun Jig Lee, J Larry Jameson
1Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.
Molecular and Cellular Biology
|September 16, 2004
Summary
Mammalian cells commonly transfer DNA enhancer elements between plasmids during co-transfection. This process, mediated by nonhomologous end-joining (NHEJ), can affect experimental results and gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Co-introduction of plasmids is standard for studying gene regulation and transfection efficiency in mammalian cells.
- Enhancer elements on plasmids are crucial for controlling gene expression levels.
Purpose of the Study:
- To investigate the phenomenon of enhancer element transfer between co-transfected plasmids in mammalian cells.
- To elucidate the molecular mechanisms underlying this observed DNA transfer.
Main Methods:
- Utilized dual-luciferase reporter assays with distinct reporter plasmids containing or lacking specific enhancer elements (e.g., estrogen response element, cytomegalovirus enhancer).
- Employed Polymerase Chain Reaction (PCR) to detect ligation of DNA fragments from separate plasmids.
- Manipulated plasmid concentration, DNA form (circular vs. linearized), and transfection methods (liposome, calcium phosphate, adenoviral).
- Introduced double-strand breaks using UV light to assess their role in the process.
Main Results:
- Demonstrated significant transfer of enhancer elements between co-transfected plasmids, altering reporter gene expression.
- Confirmed that enhancer transfer occurs only when plasmids are co-introduced, not when extracts are combined post-transfection.
- Showed increased enhancer transfer with higher plasmid concentrations and linearized DNA.
- Identified nonhomologous end-joining (NHEJ) as the mechanism responsible for ligating DNA fragments from separate plasmids.
- Observed enhanced NHEJ activity with UV-induced double-strand breaks and with liposome-mediated transfection.
Conclusions:
- Nonhomologous end-joining (NHEJ) actively mediates the transfer of regulatory DNA elements between co-introduced plasmids in mammalian cells.
- Findings necessitate caution when interpreting transfection experiments involving multiple plasmids.
- Suggests a potential mechanism for viral or exogenous DNA recombination leading to the activation of unrelated genes.