Genetic engineering is one of the most advanced branches of biotechnology. It uses modern microbiological and biochemical techniques based on genetic manipulations to solve practical problems in medicine, biology, agriculture and industry. Key procedure of genetic engineering is the construction of the recombinant DNA – recombinant DNA technology. It results in creation of DNA molecules with new primary sequencies and therefore, with new properties.
Recombinant DNA is defined as DNA molecule that contains new DNA fragments artificially incorporated into original DNA sequence.
The recombinant DNA technology resulting in production of recombinant biologically active proteins comprises several basic procedures. Among them are:
1) isolation of the DNA of interest from the host cells;
2) incorporation of the required DNA fragment into the vector;
3) the delivery of DNA into the producer cells;
4) selection of the cells that contain recombinant DNA and synthesize the required recombinant protein;
5) accumulation of the producer cells (their cloning);
6) assessment of the rate of recombinant protein synthesis, its isolation and final purification.
The process of great importance is gene cloning. At the first step of this procedure the genes, encoding necessary protein sequence, are taken from their initial DNA molecule. Also it is possible to use messenger RNA, encoding the protein of interest, which should be extracted from specific cells or tissues. In this case the primary step includes complementary DNA synthesis on RNA template by reverse transcriptase.
In order to incorporate a new gene into the recipient producer cell, it must be delivered into it by vector, or cloning vehicle. Vector contains DNA molecule that is capable of reproducing within the host cell.
A great number of natural and artificially engineered vectors are used for gene cloning. They comprise DNA molecules, plasmids, temperate bacteriophages, viruses (e.g. baculovirus or vaccinia virus), and combined vectors. The latter include cosmids, phagmids, phasmids, and others.
Cosmids contain small plasmid vector; cos-sequences of lambda-phage, responsible for DNA incorporation into phage’s head; and the large fragment (up to 30-45 kbp) for DNA of interest. Phagmids and phasmids are constructed in a similar manner, but the source of vector replication for phagmids is encoded by bacteriophage, whereas in phasmids – by plasmid sequence.
In genetic engineering of eukaryotes the method of direct microinjection of DNA into recipient nucleus is actively used now.
Two groups of specific enzymes perform the insertion of DNA into the cloning vehicle. The first group contains site-specific endonucleases or restriction enzymes (restrictases). A great variety of such enzymes was derived from different bacteria. Restrictases recognize and bind to specific base sequences in double-stranded DNA and break the phosphodiester bonds at the place of attachment.
When the DNA of the vector and the fragment of the DNA for cloning are cut with the specific restriction endonuclease and next mixed together the recombination initiates. The next enzyme, DNA-lygase, links both molecules into the continious chain; and vector recombinant hybrid molecule appears.
The vector transfers the cloning DNA into a host cell, where it should be reproduced. Frequently used prokaryotic recipient cells are E. coli, eukaryotic – yeasts fungi Saccharomyces cerevisiae, different plant cells, embryonic mammalian cells, etc.
The vehicle used for DNA delivery must carry a gene providing successful selection of the producer recipient cells that acquired the hybrid DNA. It is related with the low frequency of host cell genetic transformation. A proper marker is a gene, which codes for antibiotic resistance, thus the recipient cells maintaining functional vector DNA can be selected directly on nutrient medium with the appropriate antibiotic.
By this technique a single gene of the request from the total genome of a thousand genes can be expressed in the clone of the recipient cells.
In case of successful manipulations, the gene product (biologically active protein) will be expressed by bacterial or fungal strains and accumulated in the nutrient medium. The last point will be the indication of protein production with its subsequent purification, concentration and storage.
Genetic engineering achievements stimulated great progress in biology and medicine.
Vaccine against hepatitis B infection, based on recombinant surface viral antigen, HbsAg, was shown to be strong effective, making possible the control of the hepatitis B spread. Another recombinant anti-rabies vaccine is under clinical trial now.
Also many human recombinant proteins (hormones, enzymes, cytokines and others) were obtained by genetic engineering methods. Insulin, human growth hormone, erythropoetin, streptokinase, various kinds of interferons, interleukins, colony-stimulating factors, humanized monoclonal antibodies and many other valuable substances are now used to treat patients suffering from certain diseases. The application field of genetically engineered products is being expanded continually.