Moving genes
Adding a new gene to a cell is called transformation. The new DNA has to get into the nucleus of cell which means going past the cell membrane and cell wall that protect the cells and their contents, and finally through the nuclear membrane.
New genes can be inserted into plant, animal and bacterial cells in several different ways.
Transferring genes to bacteria
To transform bacteria such as Escherichia coli, there are two common methods – calcium chloride treatment and electroporation.
Calcium chloride: the bacteria and the bacteria with the new gene in them are placed in an ice-cold solution containing calcium chloride.
The calcium chloride changes the nature of the cell wall which allows the new DNA to enter the cells more easily. The cells are then 'heat shocked' by taking the solution containing the cells and the plasmids to 42°C for 2 minutes. Researchers usually do this by placing the tube into a waterbath. The heat shock causes the cells to take up the plasmid with the new DNA.
The cells are returned to normal growing conditions so that they can recover and the new gene begins to function within the cell.
For plants, the most commonly used method is to use the bacterium called Agrobacterium tumefaciens as a carrier of the new gene because it naturally infects plant cells. It usually infects plant cells forming crown galls which are large tumour-like swelling at the crown of the plant, just above soil level.
The new DNA is first transferred to the circular DNA molecules called plasmids which are found within bacteria. When the bacteria infects the plant cells, the new DNA enters the plant cell.
The cells are then treated to encourage them to take up new DNA. There are a few methods that can be used such as electroporation, calcium chloride treatment and biolistics.
Transferring genes to plant, animal and yeast cells
Electroporation can be used to transform cells of animals, yeast, plants and bacteria. With plant cells, they are first treated to remove their cell walls. Animal cells don’t have cell walls and don’t require this first step.
The cells are placed in a solution with the new DNA that is to be added to the cells.
The solution is then subjected to a high voltage electric shock - usually between 4 000 and 8 000 V/cm - for a fraction of a second. This causes small holes to form in the cell membrane through which the DNA enters the cells.
The cells are then placed in a nutrient solution so they can repair their membranes and cell walls and recover their normal functions.
Transferring genes to egg cells
The most commonly used method to transfer DNA directly into animal cells such as egg cells is to inject the DNA directly into a newly-fertilised egg cell using a glass capillary tube. This is called microinjection.
The egg cell containing the new DNA is implanted into a female animal. Because the gene was inserted into the DNA at the egg stage, when the cell divides, every cell in the growing embryo will contain the new DNA.
Other transformation methods
- Calcium phosphate precipitation and phagocytosis: used to transform cells of mammals. Only about 1-2 per cent of the cells are transformed so it is not a particularly efficient procedure. The DNA to be transferred is mixed with calcium phosphate. The DNA and the calcium phosphate react to form tiny grains that are solid. Cells are placed into the mixture with the grains. The cells engulf the grains in a process called phagocytosis (like amoebas eating their food). Some of the new DNA becomes a part of the cell's DNA.
- Lipofection: used to transform cells of animals, yeast, plants and bacteria. Plant cells are first treated to remove their cell walls. The DNA to be transferred is placed into liposomes which are special kinds of fats that form tiny hollow bubbles. The cells to be transformed are added to the solution containing the liposome bubbles. Since the liposomes are made up of lipids, they become part of the cell membrane of the cells and the contents - the new DNA - enters the cells.
- Biolistics: used widely in the production of genetically modified corn, and also in the genetic immunisation of animals. Tiny tungsten or gold particles are coated with the DNA to be transferred. The particles are usually about 0.004 of a millimetre in diameter. A blast of high-pressure helium gas or gunpowder shoots the particles carrying the DNA into the cells that are to be transformed.
- Viruses as carriers: used to transform cells of plants and animals but it is not a commonly used technique. A virus is chosen that will enter the cell to be transformed but not kill it. The DNA to be transferred is added to the virus DNA. The virus injects its DNA, including the new DNA, into the cell when it infects the cell.