Lab Experiment 38.3 : Transfect GFP into the COS-7 Cells using ion for Mammalian cells.
Background Information: mammalian cell membrane surface chemistry, intracellular comparatmentization and uptake mechanism is different from the prokaryotic cells or yeast. Hence specialized methods have been developed to suit mammalian cells. There are 4 major strategies to deliver the DNA in mammalian cells:
1. Chemical transfection techniques-The principle behind the chemical transfection technique is to coat or complex the DNA with a polymeric compound to a reasonable size precipitate (Figure 38.6). It facilitates the interaction of the precipitate with the plasma membrane and uptake through endocytosis. There are multiple chemical compounds have been discovered which can be able to make complex and deliver DNA into the mammalian cell.

Figure 38.6: Mechanism of chemical method mediated DNA delivery in animal cells.

Calcium Phosphate method-In this method, DNA is mixed with calcium chloride in phosphate buffer and incubated for 20mins. Afterwards, transfection mixture is added to the plate in dropwise fashion. DNA-calcium phosphate complex forms a precipitate and deposit on the cells as a uniform layer. The particulate matter is taken up by endocytosis into the internal storage of the cell. The DNA is then escapes from the precipitate and reach to nucleus through a unknown mechanism. This method suited to the cell growing in monolayer or in suspension but not for cells growing in clumps. But the technique is inconsistent and the successful transfection depends on DNA-phosphate complex particle size and which is very difficult to control.

Polyplexes method- The disadvantage of calcium phosphate method is severe physical damage to the cellular integrity due to particulate matter settling on the cell. It results in reduced cellular viability and cyto-toxicity to the cell. An alternate method was evolved where DNA was complexed with chemical agent to form soluble precipitate (polyplexes) through electrostatic interaction with DNA (Figure 38.7). A number of polycationic carbohydrate (DEAE-Dextran), positively charged cationic lipids (transfectin), polyamines (polyethylenimines) etc are used for this purpose. The soluble aggregates of DNA with polycationic complex is readily been taken up by the cell and it reaches to the nucleus for expression (Figure 21.3).