ÂThe insertion of unrelated genetic information in the form of DNA into cells called genetic transfer. The concept of gene transfer between cells first existed in bacteria; they are capable of at least four natural forms of genetic exchange. The genetic transfer technique is used very widely in basic research and applied biology. The delivery of DNA into an animal cell is a very established and fundamental procedure. It has become a very crucial tool for gene cloning, the study of gene function and the regulation and production of small amounts of recombinant proteins for analysis and verification. It helps to express the introduced genetic construct in the recipient cells or inactivate particular endogenous genes. The introduction of DNA into plants is a great agricultural potential and medical importance. Gene transfer can be transient and stable transfection. Gene transfer is one of the key purposes of the clone and it is one of the great factors in gene therapy.
There are three different types of genetic transfer methods:
1. Transfection by physical methods.
2. Transfection by biochemical methods and
3. Virus-immediately transduction.
Physical transfection methods are efficient for both in vitro and vivo gene transfer. Involvement of physical transfection works as breaching the cell membrane. It’s introducing the nucleic acid directly into the cell or nucleus.so there are advantages and disadvantages to both sets of procedures.
Chemical Transfection process must be overcome some boundaries to deliver active DNA into the nucleus. DNA transfer is the successful transfection to the nucleus, and it is the most difficult step in chemical transfection methods.
Virus-mediated transduction or bacterial transduction works as a kind of biological process that delivers bacterial DNA into host DNA. In this process, bacteriophage acts as a mediator which transfer and insert bacterial DNA into the capsid of the random selected host DNA.
This genetic transfer process can be aided by different type of injections like pronuclear injection, protoplast fusion, and ballistic DNA microinjection. DNA Microinjection is most efficient method among this category.
How Microinjection helps in Genetic transfer:
Microinjection is highly efficient at the level of individual cells. The most wonderful use of this microinjection is the introduction of DNA into the oocytes, eggs, and embryos of animals, either for generating transgenic animals or transient expression analysis. It is necessary to avoid fragmentation because DNA delivered in this manner must be very pure so it needs a lot of preparation as it is necessary. Transfection of cultured cells is automated with computer-controlled micromanipulation and microinjection processes and as well as the automated production of injection capillaries of the cell preparation procedure.
Horizontal gene transfer:
Horizontal gene transfer is also known as lateral gene transfer is made possible in large part by the existence of mobile genetic elements, such as plasmids, transposon, and bacteria-infecting viruses. In this process, newly acquired DNA is incorporated into the genome of the recipient and through either recombination or insertion. Horizontal gene transfer plays an important role in evolution and adaption in both prokaryotes and eukaryotes.
The process of genetic transfer can be explained in four key steps:
1. Isolating gene and vector:
i. Other types of vectors include modified viruses and artificial chromosomes.
ii. A vector is a DNA molecule that is used as a vehicle to carry the gene of interest into a foreign cell.
iii. These plasmids may be modified for further functionality (e.g. selection markers, reporter genes, inducible expression promoters).
iv. Bacterial plasmids are commonly used as vectors because they are capable of autonomous self-replication and expression.
v. The gene of interest can then be specifically amplified via the polymerase chain reaction (PCR).
vi. DNA can be isolated from cells by centrifugation – whereby heavier components such as nuclei are separated.
2. Digestion with Restriction Enzymes:
i. Scientists will often cleave the vector and gene with two different ‘sticky end’ restriction endonucleases (double digestion) to ensure the gene is inserted in the correct orientation and to halt the vector from re-annealing without the insertion.
ii. In order to incorporate a gene of interest into a vector, both must be cut with restriction enzymes at specific recognition sites.
iii. Restriction enzymes cleave the sugar-phosphate backbone to generate blunt ends or sticky ends.
3. Ligation of Vector and Insert:
i. DNA ligase joins the vector and gene by fusing their sugar-phosphate backbones together with a covalent phosphodiester bond.
ii. This occurs because the sticky ends of the gene and vector overlap via complementary base pairing.
iii. The gene and vector are then spliced together by the enzyme DNA ligase to form a recombinant construct.
iv. The gene of interest is inserted into a plasmid vector that has been cut with the same restriction endonucleases.
4. Selection and Expression:
i. Transgenic cells, once isolated and purified, will hopefully begin expressing the desired trait encoded by the gene of interest.
ii. This process can be achieved in a variety of ways and is called transfection (for eukaryotes) or transformation (for prokaryotes).
iii. The plasmid vector contains an antibiotic resistance gene, so only transgenic cells will grow in the presence of antibiotic.
iv. The recombinant construct (including the gene of interest) is finally introduced into an appropriate host cell or organism.
A number of clinical research successes have developed in gene transfer since 2008. Gene Transfer enables to create transgenic organisms. Transgenic animals can reduce the high demand of product quantitatively and qualitatively so it can be concluded that gene transfer process will help in future demands and supply undoubtedly.