Transgenic animals: what are they for? Prospects for the creation and use of transgenic animals with new beneficial properties

Transgenic animals, examples of which will be given below, were obtained experimentally. They contain in all their cells an additional DNA transgene introduced into the cells of an animal with chromosomes and realized there. A transgene is inherited according to the laws of Mendel.

Reasons for creating

Transgenic plants and animals vary in the degree of complexity of their creation. Animals are used to solve theoretical problems in biomedicine and agriculture. Among the reasons for the creation of transgenic organisms are the following:

• animals with the gene for growth hormone under equal conditions give increased growth rates;
• to enhance immunity to infections;
• for biologically active substances;
• to obtain human protein from transgenic animals;
• the use of animals as bioreactors for obtaining medications.

The first such animals were obtained in 1974 in Cambridge by a scientist named Rudolph Janisch. He introduced the monkey DNA virus into the mouse embryo. In Russia, the first transgenic animals were bred in 1982.

Getting

Why are transgenic animals needed? Their creation occurs by transferring cloned DNA to the nuclei of fertilized eggs called zygotes, or stem embryonic cells. After that, altered zygotes or eggs are transplanted into reproductive organs.

If you do not go into details, then the whole process is as follows:

1. The cloned gene is introduced into the nucleus of the egg, previously fertilized.
2. Fertilized eggs with embedded DNA are placed in a pre-selected female for further gestation of the transgenic fetus.
3. The selection of offspring born through the introduction of foreign DNA is carried out in such a way that the cloned gene is contained in each cell of the resulting organism.
4. Animals that carry a cloned gene in germline cells are crossed.
5. Thus, a new genetic line is obtained.

Such experiments are very expensive in terms of the time necessary for this. Despite this, transgenesis is becoming a powerful tool for the study of human diseases. Using transgenesis, work is underway on the genetic modification of the mammary glands of animals to produce protein identical to human from milk, as well as for the manufacture of pharmaceutical preparations.

Animal transgenesis methods

What are they needed for? Transgenic animals are individual individuals whose genome is distinguished by artificially supplemented genetic information. The transgene is in one case an independent DNA region with its own regulatory sequences. In another, it is a DNA hybrid (recombinant) gene created from various molecules.

A transgene is an alien gene introduced artificially and fixed in the animal’s DNA. And transgenesis is the process of integration and transfer of foreign genetic information into the animal’s genome.

There are certain methods for producing transgenic animals. Among them are:

1. Microinjection method.
2. The method of using retroviral vectors.
3. Transplantation of nuclei of cells that are cultured in vitro.
4. Method of liposomes - DNA carriers.
5. The method of using germ cells of the testes.

Microinjection method

For the first time, the microinjection method was used to obtain transgenic animals. This happened in 1985 at the same time in two research laboratories. One of them belonged to the USA, the other was in Germany. This method is currently the most commonly used.

The microinjection method is based on the introduction of a solution of gene constructs into the male pronucleus (nucleus precursor) of zygotes. During fertilization, two nuclei are formed in the egg, one of which is male (male pronucleus), the other is female (female pronucleus). Male and female pronuclei after convergence are combined into a common nucleus with the connection of paternal and maternal chromosomes into one embryo genotype.

The success of microinjection can be judged by an increase in pronucleus volume of 1.5 times. When the procedure is completed, the resulting embryos are cultured for several hours, and then several females are planted in the uterus simultaneously.

After the birth of the offspring, an analysis is made for the integration of the transgene by sampling. The number of transgenic babies of the total number of offspring born using this method is very small. In test mice this is approximately 15%, in pigs from 10 to 15%, in sheep, goats and cattle this indicator is tacked from 5 to 10%. To improve this ratio, scientists are working to this day.

Method of using retroviral vectors

Transfer of DNA into the embryonic lines of animals using retroviral vectors is a rather effective method. Retroviruses are nucleated viruses. Their genetic material is single stranded ribonucleic acid (RNA).

Retroviruses can be ecotropic, that is, able to reproduce in the cells of one animal species or several closely related species. For example, the mouse leukemia virus can multiply only in mouse or rat cells. Amphotropic viruses are capable of spreading in many animal species.

The retrovirus enters the cell with the help of micropinocytosis - the transport of small molecules. In some viruses, transcription occurs in the cell nucleus, in others in the cytoplasm. If integration occurs in the genome of generative cells, then the retroviruses are inherited. Here we are talking about endogenous retroviruses.

Viral DNA was first detected in adult mouse cells in 1974. Infection of zygotes in most cases leads to the production of transgenic animals, since integration is possible if the cell enters into indirect division after DNA replication.

Benefits of using retroviral vectors:

1. Nearly 100% of retrovirus-treated embryos can be infected.
2. It becomes possible to carry out gene therapy of hereditary diseases.
3. Retroviruses make possible the transformation of individual organs (for example, mammary glands to increase milk yield).

Disadvantages of using retroviral vectors:

1. Limited capacity.
2. Inhibition of transgene expression in vivo.
3. Low titer (solved by pseudotyping).
4. Possibility of cellular oncogenes.
5. The inability to inherit the transformation of individual organs.

Cell nucleus transplantation method

A feature of the method is the use of transformed cell lines to obtain transgenic individuals using gene constructs. For this, stem lines are taken, as well as somatic cells that are cultured in vitro, that is, in vitro.

One of the methods of nuclear transplantation is the method of micromanipulation, and it consists in the following:

1. With the help of a micropipette, pronuclei are extracted after the puncturing of the zones of the pellucide and the zones of the plasma membrane has occurred.
2. With a large diameter pipette, a nucleus with a complete set of chromosomes from the donor is introduced into the same puncture.

Using this method, the zygote cytoplasm is injured much less, like the donor nucleus.

There is another way to transplant a nucleus. This method consists in using cytochalazines, that is, substances that are synthesized using fungi, for this purpose. The structure of microenzymes (filaments consisting of molecules of the globular actin protein) is destroyed by cytochalasin B, after which the nucleus can be located in a unique way.

The nucleus, using a thin strand of cytoplasm, remains connected to the cell. Then this bond is artificially broken by centrifugation. Cytoplasts (cells without a nucleus) and karyoplasts (nuclei surrounded by a cytoplasm) are formed. In a density gradient, cytoplasts separate from intact cells, and can be used to connect other cells with karyoplasts to make a viable cell.

Method of liposomes - DNA carriers

This is the most harmless method of transgenesis. Its feature is the use of liposomes for the conservation and transfer of genes in them. Liposomes are created with various properties. They are not immunogens and do not have toxicity.

However, this method is not effective because the DNA transported in this way into the cell is immediately captured by the lysosomes and destroyed by them.

The method of using germ cells of the testes

Sperm is a natural vector that transports DNA into a cell. But the studies conducted in this area have shown mixed results of the method. In the experiments of M. Lavitrano in 1989, 30% of the experimental mice that were obtained by injecting the modified sperm into the unfertilized mouse eggs turned out to be transgenic and could inherit the transgene.

In another case, many attempts to repeat this result in other laboratories were unsuccessful; under similar conditions, different results were obtained. From all this, it can be assumed that a successful transformation of DNA is observed only at a certain stage of the cell cycle. Studies are still ongoing to determine the mechanism for the incorporation of exogenous DNA into the sperm genome.

The possibility of using sperm after cryopreservation is a positive aspect of this method. The negative side is considered a large number of micromanipulations required during transgenesis using this method.

But still, sperm have the ability to absorb DNA and accumulate it in the nucleus. To achieve a greater effect of combining DNA with a sperm, there are several methods: liposome, injection into the testes, into the seminiferous tubules by creating pores in the lipid membrane under the influence of an electric field.

Genes Off

Gene shutdown is a suppression of gene expression, i.e. gene silencing or gene knockout. In this process, the expression of the required gene is terminated, but the nucleotide sequence remains unchanged.

Gene shutdown can occur when information is transferred from DNA to RNA, that is, transcription, and after it. Gene shutdown mechanisms protect the body from viruses and jumping genes. Therefore, silencing is part of the immune system that protects against foreign DNA. Transgenic animals with turned off genes and their creation are called gene targeting.

Gene inheritance according to Mendel’s laws

Mendel’s laws describe the characteristics of the transmission of hereditary characteristics from the body of the parent to the body of the cub. These principles are the basis of classical genetics. Basically, all scientific articles reveal one of Mendel’s rules and a couple of laws.

The rule of the first generation of hybrids and their uniformity says that these hybrids always inherit one of the characteristics of the parent.

The law of the splitting of signs is Mendel’s first law. It says that ¾ experimental animals showed dominant signs, and ¼ recessive ones.

The law of independent inheritance of characters is the second law. Here, Mendel describes the distribution of characters when crossing polyhybrid and dihybrid.

Beneficial features

The creation of transgenic animals with new beneficial properties is an ongoing process.

Transgenic animals, examples of which are given below, already exist in the modern world:

• In Great Britain, sheep were bred using transgenesis. The milk of these animals contains a factor that promotes blood coagulation.

• In transgenic pigs created in Russia, the metabolism was changed by introducing a somatotropin gene, which allowed to reduce the fat content of meat.
• Americans are working to create cows whose milk contains human albumin, which is used to maintain normal blood pressure.

Prospects for the use of transgenic animals

In the near future, it is planned to create such animals, some genes of which are knocked out, while others are introduced into their genome.

• Getting modified milk.

It is assumed that such milk in its composition will be as close as possible to the composition of human mother's milk. In this direction, work is being done with embryonic stem cells.

• The use of transgenic animals to obtain organs that are transplanted to humans.

It is no secret that humanity needs donor organs. Now geneticists are working on the cultivation of such organs in the body of animals. For example, the organs of pigs could well fit in their size and composition. But they will be immediately rejected by the human immune system. To prevent this from happening, a transgenic pig is created, in which the histocompatibility genes are turned off, and human histocompatibility genes are introduced instead.

• Cloning of transgenic animals.

Creating transgenic animals is a labor-intensive process. According to statistics, less than 50% of individuals expressing a transgenic protein account for 100 injected sheep zygotes, 40 mouse zygotes, and 1500 cow zygotes. With a successful attempt to obtain a transgenic animal, it is not at all necessary that a transgene will be inherited by its descendants. Therefore, cloning an animal with the necessary genetic parameters is an optimal way out.