3.3. Genetic engineering

Genetic engineering is a system of experimental methods which allow constructing artificial genetic structures in the form of recombinant or hybrid DNA molecules at laboratories (in vitro).

Recombinant DNA technology uses the following methods:

1. Specific DNA splitting by restrictases. Targets for restrictases are restriction sites. One type of restrictases make cuts along the symmetry axis forming so called blunt ends. Others make cuts with a turn from symmetry axis forming sticky ends that is fragments which have single strand mutually complimentary parts with the length of 4 nucleotides at their ends. Such fragments are very convenient to form recombinant DNA.

2. Nucleotide sequencing. DNA fragments, which differ in the size, could be separated by electrophoresis and investigated. It allows building a restriction map which indicates the position of each restriction site.

3. Construction of recombinant DNA:

To receive a recombinant DNA, plasmids should be isolated from E. coli. After that part of circular DNA of the molecule is deleted. This is done with the help of restrictase. The DNA fragment which was chosen for translocation is created by the same restrictase. DNA fragment (gene) is mixed with plasmid and they are connected with the participation of ligase. This is recombinant DNA.

The following variants are possible:

a. splicing of homogenous sticky ends (restrictase-ligase method). Complimentary parts tend to association due to pairing of bases. To restore damages an enzyme DNA-ligase is used.

b. splicing of blunt ends (connector method). Blunt ends could be connected by DNA-ligase. Effectivity of this reaction is lower than at splicing of sticky ends.

c. splicing of heterogenous sticky ends. Linkers are used in this case. Linkers are chemically synthesized oligonucleotides which are restriction sites or their combination. There are linkers “blunt end- sticky end”.

This procedure is complex and allows receiving only small amounts of recombinant DNA.

4. Cloning of recombinant DNA:

a. DNA cloning in vivo. If we add recombinant plasmids to the culture of E. coli, then they could be included into bacterial cells. During reproduction of the recombinant bacteria, the newly formed cells also contain recombinant plasmids. It is possible to isolate DNA from them.

b. amplification of DNA in vitro. It is a laboratory method for creating multiple copies of small segments of DNA.

This method was developed in 1985 by Kary Mullis and received the name polymerase chain reaction (PCR). In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR. The excessive amount of 2 synthetic primers is added to analyzed sample of DNA. Primers are oriented in such a way that the synthesis with the help of polymerase goes on only between them. The number of copies of this DNA part is doubled. Amplified part is called an amplicon. Amplification is repeating cycles which form a three stage process: 1) DNA denaturation at 95 degrees; 2) annealing of primers with the complimentary sequences (40-60 degrees); 3) completion of polynucleotide chains with the help of DNA-polymerase at temperature of 70-75 degrees.

Fig 8. Amplification of DNA in vitro

The continuation of one cycle is less than 3 minutes. Thus for 2 hours about one billion of copies of the DNA determined sequence could be received. PCR is sometimes called cell-free molecular cloning.

New diagnostic methods are created with the help of PCR. They are used to find genetic and infectious diseases. This method is also used to find AIDS at early stages. Method to analyze individual sperm is used in forensic medicine.

5. Gene introduction into the cell. Gene should be introduced into a cell so that it is not damaged by cell nucleases but it should be integrated into the cell genome. Two methods are used:

1. Transduction is gene introduction by using vectors. Bacterial plasmids and viruses are usually used as vectors. The aim of a vector is to carry the chosen DNA to the recipient cell and build it into the genome.

There should be a marked gene in the structure of a vector. It allows to select changed cells. There could be 2 groups of marked genes:

- selective genes, which are responsible for sensibility for antibiotics or herbicides;

- reporter genes, which code for neutral cell proteins the presence of which could be easily detectable in tissues.

Regulatory sequences which are responsible for gene expression should be also built into a vector molecule.

Types of vectors:

Bacterial plasmids. One of the most widely used plasmids is pBR 322 is created on the basis of plasmids isolated from E. coli;

Viruses. There are viruses which do not lead to cell death but they are bult in the host-cell genome and they are reproduced together with their host-cell. They can also lead to uncontrolled growth of the cell, thus they transform the cell into onco-cell.

Hybrid vectors. They contain DNA of phage and plasmid: cosmid and phasmid.

2. Transformation is direct gene introduction into the cell. There are the following types of transformation.

Transfection. DNA is adsorbed on the crystals of calcium phosphate. They are incorporated by the cell due to phagocytosis.

Micro-injections with DNA with the help of micro-pipettes with the diameter about 0.1-0.5 microns and micro-manipulator.

Electroporation is based on the fact that impulses of high voltage reversibly increase permeability of bio membranes.

“Mini cells” are received due to blocking of donor cells in mitosis by colcemide. Around each chromosome a new nucleus membrane is formed. Then they are worked over by cytochalasine B and are centrifuged. Mini cells are formed which are also called micro nuclei. They are incapsulated in cytoplasmic membrane. Special conditions are chosen for their splicing.

Folding into liposomes is used to protect exogene material from damage by restrictases.

Biological ballistics method is one of the most effective for plant transformation. Small particles of wolfram receive DNA vector. They are placed inside biolistic cannon. After that they are fired with the high rate and breaking cell walls they enter cytoplasm and nucleus.

With the development of genetic engineering there is a possibility to make micro-organisms synthesize some substances which are very difficult to recieve by other methods: interpheron, insulin, somatostatin, somatotropin, urokinase enzyme, some factors for blood coagulation, etc. all these proteins are used to cure different diseases.

Plasmids could be introduced in to eukaryote cells. Genetic transformation of mammal somatic cells allows to study the regulation mechanisms of gene expression and modify the cell genetic apparatus which have importance for medical genetics. Cultures of transformed mammal cells are used to receive different substances.

Gene therapy is disease treatment with genes. There are 2 types of gene therapy.

Substitution gene therapy is introduction of normal gene into the cell.

At correctional gene therapy there is an exchange of faulty gene by a normal as a result of recombination. This method is being tested only at laboratories.

Gene therapy is used to correct violations at Duchenne muscular dystrophy, mucoviscidosis (hereditary severe disease of lungs). Hereditary diseases which are supposed to be treated in the future with genes are arthritis, phenylketonuria, etc.

Gene therapy could not be used only for hereditary diseases. The problem of acquired immunodeficiency syndrom (AIDS) could be solved if scientists could find new genes which introduction into lymphocytes with HIV (human immunodeficiency virus) could stop the further development of the virus.

Transgenic animals. To change the properties of the whole organism scientists need to change genome of sex cells. Nowadays there are methods to introduce genes in fetus cells at mammals, flies and some plants. Microinjection of clone genes is done into the fertilized ovum then it is implanted into mother’s body. Gene could be introduced into sperm and then fertilization could be made by them.

In England there are transgenic sheep which milk has blood coagulation factors. There is a transgenic cattle which has human albumin in its milk. Each dairy cow can produce 80 kg of recombinant human albumin per year. Transgenic animals are used for organ transplantation. One of the favorite organ donors are pigs, since there they similarity of the anatomical and immunological properties. One of the signals the body to attack a foreign body are proteins that are localized on the outer surface of the membrane. In transgenic pigs, these proteins are replaced by human ones.