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Published on: September 10, 2016
Regulation of G418 selection efficiency by cell-cell interaction in transfection.
K Sasaki1, H Mizusawa, M Ishidate
1Department of Cell Biology, Hatano Research Institute, Food and Drug Safety Center (FDSC), Kanagawa, Japan.
Somatic Cell and Molecular Genetics
|November 1, 1992
Summary
Optimizing calcium phosphate (CaPO4) transfection requires careful consideration of culture conditions. Cell-cell contact significantly impacts G418 resistant colony formation, potentially masking true transfection efficiency.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- Calcium phosphate (CaPO4) precipitation is a common method for DNA transfection into mammalian cells.
- The pSV2-neo DNA construct confers resistance to the antibiotic G418.
- Optimizing transfection protocols is crucial for efficient gene delivery and selection.
Purpose of the Study:
- To determine the optimal conditions for calcium phosphate (CaPO4) mediated DNA transfection using pSV2-neo.
- To investigate the influence of culture parameters on the induction of G418 resistant colonies.
- To understand the role of cell-cell contact in G418 resistant colony formation.
Main Methods:
- Transfection of BALB 3T3 cells with pSV2-neo DNA using CaPO4 precipitation.
- Enumeration of G418 resistant (G418r) colonies under varying conditions.
- Derivation and coculture of G418r clones with G418 sensitive (G418s) parent cells.
- Analysis of cell-cell contact effects on colony formation.
Main Results:
- Amount and size of carrier DNA, cell plating density, precipitate treatment time, and G418 expression time significantly affect G418r colony induction.
- Coculture of G418r clones with G418s cells showed decreased colony formation with increasing G418s cell numbers.
- Cell-cell contact is necessary for reducing G418r colony formation, likely via contact-mediated cell killing, not gap junctions.
- High-density plating, prolonged precipitate treatment, and extended G418 selection can mask transfection efficiency.
Conclusions:
- Culture conditions, particularly cell density and contact, critically influence the observed transfection efficiency.
- Contact-mediated cell killing can reduce the number of G418 resistant colonies.
- Transfection efficiency assessed by G418 resistance requires careful evaluation of culture parameters to avoid masking effects.
