Genetic engineering and its principles, gene transfer

Principles of Genetic Engineering:-

1. Introduction:-

> The technology of recombinant DNA was developed in 1973 by Boyer and Cohen.

> It is popularly known as genetic engineering. 

> Recombinat DNA:- When foreign gene is inserted into a vector, then it is called as recombinant DNA.

> Objective:- This is the natural mathod of amplification of gene of interest.

2. Process of Recombinant DNA Technology:- The complete process of recombinant DNA technology includes multiple steps-

Step-1. Isolation of Genetic Material:- The first and the initial step in Recombinant DNA technology is to isolate the desired DNA in its pure form i.e. free from other macromolecules.

Step-2. Cutting the gene at the recognition sites:- The restriction enzymes play a major role in determining the location at which the desired gene is inserted into the vector genome. These reactions are called ‘restriction enzyme digestions’.

Step-3. Ligation of DNA Molecules:- In this step of Ligation, the joining of the two pieces – a cut fragment of DNA and the vector together with the help of the enzyme DNA ligase.

Step-4. Insertion of Recombinant DNA Into Host:- In this step, the recombinant DNA is introduced into a recipient host cell. This process is termed as Transformation. 

Step-5. Amplifying the gene copies:- Once the recombinant DNA is inserted into the host cell, it gets multiplied. As a result the inserted gene of interest is also multiplied.

Gene Transfer:- 2 types

A. Direct Gene Transfer (Vectorless gene transfer)

B. Indirect Gene Transfer (Vector mediated gene transfer)

Direct Gene Transfer (Vectorless gene transfer):- When stable transformation is achieved by incorporating the desired gene into the genome of plant cells without the help of any biological factor, it is called direct gene transfer.

1. Partical gun method:-

•      It is a physical method of gene transfer.

•      It is also called Biolistic method.

•      In this, tungsten or gold particles of 1-2µm diameter are coated with DNA and fired them into the cell by a particle gun.

•     Gold is expensive, but it is less harmful to cells.

•     The cost of a particle gun is almost 15 lakhs.

•     The particles are accelerated by compressed helium gas in the particle gun.

•      DNA reaches inside the nucleus and becomes incorporated into the plant's genome.

•     By this method, the gene is transferred to the meristem and embryo.

2. Liposome-mediated gene transfer:-

•   Liposomes were discovered in the year 1960 by British haematologist “Dr.Alec D.Bangham”. 

•   These are the concentric bilayered vesicles in which an aqueous core is entirely enclosed by a membrane lipid bilayer mainly composed of natural and synthetic phospholipids. 

•   Its Size range varies from 20nm - 3μm microns. 

•   Liposomes are spheres of lipids which can be used to transport the molecules into the cells. 

•   Liposomes deliver not only nucleic acids, but also other targeting ligands, like peptides, antibodies, aptamers, folic acid. 

•   Chemical modifications of liposome can efficiently improve their performance.

3. Chemical method:-

•      It is also called as PEG mediated Gene Transfer.

•      In this method gene transfer is induced by PEG. (PEG = Poly Ethylene Glycol)

•      Transformation medium is added in a beaker that has a high concentration of Mg2 + ions.

•      Now plant protoplasts are put into this nutritive medium.

•      Now put the desired gene in the beaker.

•      Now take the pH to 8.

•      Now add 20% PEG.

•      Now decrease the concentration of PEG and increase the concentration of Ca2+ ions.

•     Now incubate for some time.

•     The transformation frequency increases 10 to 1000 times when plant protoplasts are transferred to ice after heat-shock at 45°C for 5 minutes just before adding DNA.

B. Indirect Gene Transfer (Vector mediated gene transfer):- When stable transformation is achieved by incorporating genes into the genome of plant cells with the help of a biological factor, it is called indirect gene transfer. Agrobacterium bacterium is used in this.

Agrobacterium mediated Gene Transfer:-

•  Agrobacterium is a gram -ve bacterium found in soil around plant roots.

•  Two species of Agrobacterium bacteria cause diseases in dicot plants:-

i. Agrobacterium tumefaciens

ii. Agrobacterium rhizogenes

i. Agrobacterium tumefaciens:- It causes Crown gall disease in angiosperm plants. Ti-plasmid is found in it.

ii. Agrobacterium rhizogenes:- It cause hairy root disease in angiosperm plants. Ri - plasmid is found in it.

•  Recombinant DNA is made by incorporating the desired gene into the T - DNA portion of the Ti plasmid. Which are transferred to Agrobacterium cell.

•  Now cut round pieces of sterilized leaf.

•  Now incubate these pieces with Agrobacterium overnight.

•  Now kept these pieces in Shooting medium for 2 days.

•  Now add Kanamycin and Carbenicillin in this shooting medium and cultured for 3-4 weeks.

•  Now transfer this to the rooting medium, add Kanamycin and Carbenicilin, culture it for 3-4 weeks.

•  Now establish this plant in a pot in green house.

•  After a few days, establish the plant in the soil of the field.

Mechanism of Gene Transfer:-

•  The T - DNA portion of the Ti - plasmid has the property that it can integrate into the plant's genome.

•  Leaf pieces release the Aceto - Syringine chemical that activates the Vir - Operon of T - DNA.

•  Once activated, the T-DNA portion separates from the plasmid and enters into many plant cells.

•  Now this T-DNA integrates into the genome by going into the nucleus.

•  This results in stable transformation of the plant.