ADVERTISEMENTS:
In this article we will discuss about the top two techniques used for introduction of foreign DNA without vectors. The techniques are: 1. Electroporation Technique 2. Microinjection Technique
ADVERTISEMENTS:
Some direct methods have also been developed for transferring DNA of one organism to a recipient without the participation of a vector.
Two such methods are:
ADVERTISEMENTS:
1. Electroporation technique and
2. Microinjection technique.
The principles of these techniques are briefly discussed below:
1. Electroporation Technique:
ADVERTISEMENTS:
When biological cells are exposed to a brief pulse of high voltage electric current, minute pores are formed in the cell membrane. The pores are large enough to allow passage of DNA molecules. In case of plant and bacterial cells, the cell wall needs to be enzymatically removed and the resultant protoplasts are subjected to electroporation technique.
For electroporation treatment, the recipients are placed in a solution containing a high concentration of the DNA sample to be introduced. The suspension is then exposed briefly to an electric current of 200 to 600 volts per cm, depending on the recipient. Some DNA molecules pass through the membrane pores created by the high-voltage electric current into the recipient cells.
The incoming DNA molecules may eventually recombine with the cell DNA to produce recombinants. By proper selection, it is possible to generate transgenic cells by this technique. Such transgenic cells can be regenerated into whole individuals by adopting the conventional tissue culture methods in case of plants.
2. Microinjection Technique:
One of the surest methods of introducing foreign DNA into a recipient cell is by direct injection of DNA molecules. This method is specially suitable for animal cells. The foreign DNA can be introduced into a cell by puncturing the cell membrane with a capillary micropipette having a fine tip. The tip should be much smaller in diameter than that of the recipient cell. The introduced DNA, if it survives in the recipient, may eventually recombine with the host DNA to give rise to a recombinant.
A variation of the microinjection technique is to introduce DNA by bombardment of the recipient cells with micro projectiles coated with DNA. The bombardment is carried out with the help of a gene- gun. The micro projectiles are minute beads of 1 μm diameter made of tungsten or gold. The beads are coated with the DNA to be introduced by allowing DNA to be precipitated on them. These DNA-coated beads are fired from a gene-gun with the help of a burst of helium gas at a velocity of about 430 meters per second on a suspension of the target cells.
This method of DNA injection via micro projectiles is specially applicable for plant cells with a cellulose wall. The projectiles penetrate through the wall carrying with them the DNA into the cells. The cells in direct line of bombardment are often killed, but some cells may survive the impact and give rise to transgenic cells through recombination of the incoming DNA and the cell’s genome. From the transgenic cells whole plants can be regenerated. Bombardment technique has been successfully used in maize (Zea mays) to generate transgenic plants which are resistant to the herbicide phosphoinothricin.