Introducing Alien DNA into Host Cell | Nucleic Acid | Plants | Genetics

The following points highlight the three methods to introduce alien DNA into host cell. The methods are:- 1. Microinjection 2. Electroporation 3. Shot-Gun/Gene Gun Method of DNA Introduction.  

Method # 1. Microinjection:

DNA can also be stably introduced into tissue culture cells by its direct microinjection into the nuclei of the cells, using a glass micropipette that has been drawn out to an extremely thin diameter (from 0.1 to 0.5 microns). Such a procedure requires some fairly sophisticated equipment (a micropipette puller for making the needles, and a micromanipulator to position the needles correctly for injections); but given this equipment and enough practice, one can inject 500 to 1000 cells per hour with DNA, and have up to 50 per cent of the injected cells stably integrate and express the injected genes. It is found that injected DNA will integrate at random into the nuclear DNA, and if an injected gene is attached to a suitable promoter it might be expressed.

The advantage of this procedure is that in principle any piece of DNA can be introduced into any cell; no selective pressure needs to be applied to maintain the transferred gene. This method has been used to transfer the gene for rat growth hormone into mice, in a few of which the gene was expressed, resulting in the production of “giant” mice. The same procedure of introducing pieces of DNA into plant cells is also followed.

The disadvantage of microinjection is the expensive equipment that is required, the extensive practice needed to master this tedious technique, and the relatively small number of cells that can be treated in one experiment.

Also after microinjection, the egg must be re-implanted in a surrogate mother, and only after gestation can the progeny be screened for expression and correct regulation of the foreign DNA. This is therefore a slow, labour-intensive method of genetic manipulation, and the small size of population which can be produced for screening inevitably reduces the chances of success.

Method # 2. Electroporation:

This method is based on the use of short electrical impulses of high field strength. These impulses increase the permeability of protoplast membrane and facilitate entry of DNA molecules into the cells, if the DNA is in direct contact with the membrane.

In view of this, for delivery of DNA to protoplasts, electroporation is one of the several routine techniques for efficient transformation. However, since regeneration from protoplasts is not always possible, cultured cells or tissue explants are often used. Hence, it is important to test whether electroporation could transfer genes into walled cells. In most of these cases no proof of transformation was available.

The electroporation pulse is generated by discharging a capacitor across the electrodes in a specially designed electroporation chamber. Protoplasts in an ionic solution containing the vector DNA, are suspended between the electrodes, electroporated and then plated as usual.

Transformed colonies are selected. Using electroporation method, successful transfer of genes was achieved with the protoplasts of Petunia, maize, rice, wheat and sorghum.

Transformation frequencies can be further improved by:

(i) Using field strength of 1.25kV/cm,

(ii) Adding PEG after adding DNA,

(iii) Heat shocking protoplasts at 45 ‘c for 5 minutes before adding DNA and

(iv) By using linear instead of circular DNA.

Method # 3. Shot-Gun/Gene Gun Method of DNA Introduction:

In recent years, it has been shown that DNA delivery to plant cells is also possible, when heavy metallic pellets (tungsten or gold) coated with the DNA of interest are accelerated to a very high initial velocity (1,400 ft/sec.)

These microprojectiles, normally 1-3 nm m diameter, are carried by a macroprojectile’ or the “bullet” and accelerated into living plant cells (target cells can be pollen, cultured cells, cells in differentiated tissue and meristems) so that they can penetrate cells walls of intact tissue.

The acceleration is achieved either by an explosive charge (cordite explosion) or by using shock waves initiated by a high-voltage electric discharge. The design of two particle guns used for acceleration of microprojectiles are shown in Fig. 11.17.

The advantages of this method over microinjection include:

(i) Thousands of particles are accelerated at the same time, causing multiple hits resulting m transfer of genes into many cells simultaneously;

(ii) Since in act cells can be used, some of the difficulties encounter with the use of protoplasts are automatically circumvented;

(iii) The method is universal in is application, so that cell type, size and shape or the presence/absence of cell walls do not significantly alter its effectiveness. In view of this, particle bombardment method using microprojectiles has a great promise in a variety of plant species, particularly the cereals.