VPB 321: Animal Biotechnology (2+1)

METHODS

• Various methods are used to introduce foreign DNA into a eukaryotic cell. Several materials are used as carriers for transfection.
  • Calcium Phophate method : One of the cheapest methods of transfection is by calcium phosphate, discovered by F. L Graham and A.J. van der Eb in 1973. HEPES-buffered saline solution containing phosphate ions is combined with a calcium chloride solution containing the DNA to be transfected. Mixing of these two results in precipitate due to binding of positively charged calcium and the negatively charged phosphate. This suspension of precipitate is then added to the cells to be transfected, usually a cell culture grown in a monolayer. The cells take up some of the precipitate and  the DNA.
  • Liposome method : It is a very efficient method of transfection. Liposomes, a small, membrane-bounded body that are in similar to the structure of a cell and can actually fuse with the cell membrane. The DNA to be transfected is trapped into the liposomes. This DNA loaded liposome is added to the cells to be transfected and release the DNA into the cell.
  • Cationic polymers : Cationic polymers such as DEAE-dextran or polyethylenimine. The polycation binds to the negatively charged DNA and the complex is then taken up by the cell via endocytosis.
  • Gene gun transfection – It is a method where the DNA is coupled to a nanoparticle such as gold which is then shot directly into the target cell’s nucleus.
  • Optical transfection - It is a method where a tiny hole is generated in the plasma membrane of the target cell using LASER. In this method, single cell at a time is treated and making it particularly useful for single cell analysis.
  • Other Substances - Nucleofectin, lipofectin are also used to transfect nucleic acids into the target cells.