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Related Experiment Videos

Optimizing conditions for calcium phosphate mediated transient transfection.

Ling Guo1, Liyang Wang1, Ronghua Yang1

  • 1College of Life Science, Shaanxi Normal University, Xi'an 710062, China.

Saudi Journal of Biological Sciences
|April 8, 2017
PubMed
Summary

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Optimizing calcium phosphate DNA transfection, this study details key factors for enhanced efficiency in difficult-to-transfect cells. Key improvements include specific buffer systems, media, and glycerol shock treatments for better DNA delivery.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biotechnology

Background:

  • Calcium phosphate (CaP) mediated transfection is a cost-effective method for DNA delivery into mammalian cells, established for over 30 years.
  • Low transfection efficiency in highly differentiated and non-tumor cell lines, such as Chinese Hamster Ovary (CHO) and C2C12 myoblasts, limits its broader research application.

Purpose of the Study:

  • To systematically optimize calcium phosphate mediated DNA transfection efficiency.
  • To identify critical parameters for improving DNA delivery in challenging cell types like CHO and C2C12 cells.

Main Methods:

  • Systematic evaluation of factors influencing transfection efficiency, categorized into calcium phosphate-DNA co-precipitation and on-cell treatments.
  • Optimization of buffer systems (HBS at pH 7.10), transfection media (DMEM with FBS), particle concentration, intermittent vortexing, and glycerol shock treatment duration.
Keywords:
C2C12, mouse myoblast cellsCHO, Chinese hamster ovary cellsCalcium phosphate transfectionCo-precipitationFBS, fetal bovine serumIntDen, integrated densityPEG, polyethylene glycolPen-Strep, penicillinstreptomycinTransfection efficiency

Related Experiment Videos

  • Incubation time and glycerol shock application timing were critical parameters investigated.

    • Main Results:

    • HBS buffer at pH 7.10 significantly improved transfection rates compared to HEPES buffer in CHO cells.
    • Fetal Bovine Serum (FBS) in transfection medium and a higher concentration of co-precipitated particles with intermittent vortexing were crucial for efficiency.
    • Glycerol shock treatment after 6-hour incubation yielded the highest transfection efficiency in both CHO and C2C12 cells.

    Conclusions:

    • Specific conditions, including HBS-buffered saline, optimized pH, glycerol shock, vortexing, transfection medium composition, and particle concentration, are essential for enhancing calcium phosphate transfection.
    • This optimized protocol significantly improves DNA delivery efficiency in highly differentiated cells, expanding the utility of this cost-effective method.