Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Mammalian cell transfection is crucial for gene function studies.
• Existing methods like microinjection have limitations.
• Optimizing DNA delivery to the nucleus is key for efficient transfection.

Purpose of the Study:

• To develop a highly efficient, reproducible transfection procedure for mammalian cells.
• To compare the new method with manual microinjection.
• To identify factors influencing transfection efficiency.

Main Methods:

• Developed a polyethylene glycol (PEG)-based shock method for cells in suspension.
• Transfected cells 4 hours post-replating.
• Utilized recombinant plasmids with simian virus 40 A or herpes simplex virus thymidine kinase genes.
• Compared results with manual DNA microinjection into cell nuclei.

Main Results:

• Achieved up to 70% transfection efficiency in specific cell lines (tk-ts13).
• Transfection efficiency is highly dependent on the cell line used.
• PEG method is simpler and more reproducible than microinjection.
• Enzymatic function (thymidine kinase activity) improved positive transfectant percentage compared to immunofluorescence.

Conclusions:

• The PEG-based transfection procedure offers high efficiency and reproducibility for low-molecular-weight DNA.
• DNA delivery to the nucleus is a critical factor in transfection success.
• This method is suitable for phenotypic and genotypic studies requiring gene transfer into numerous cells.