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Molecular biology

A factory of mice

Transgenic rodents produce the protein responsible for blood coagulation

UNIFESPMice embryosUNIFESP

Producing cows and goats that have in their milk the human protein responsible for blood coagulation, called the IX factor, is going to represent a major advance for people suffering from hemophilia. On being isolated from the milk, this protein can be transformed into a cheaper and more easily available product for these sick patients who today control their illness with medicines derived from healthy blood donors. Hemophilia is a genetic illness characterized by problems in blood coagulation and without the IX factor, the patients can suffer hemorrhaging from any skin cut, for example. Studies for this new possibility of hemophilia treatment were indicated by way of a partnership between researchers from the Federal University of Sao Paulo (UNIFESP) and the Brazilian Agricultural Research Corporation (Embrapa). By making use of molecular biology resources, the researchers from these two institutions are working to develop genetically modified cows and goats capable of producing the IX factor in their milk.

The first step in creating these “bio-factories” was to develop at UNIFESP a strain of transgenic mice that possess in their genetic code the codifying gene of this human protein. Milk samples of the transgenic rodents containing the coagulation protein are being evaluated by a team from the Support Hospital of Brasilia. “We’re collecting blood from patients with hemophilia to evaluate, in vitro, the coagulation effect and to compare it with today’s products’, says the researcher Elíbio Rech, the project’s coordinator from the Brazilian Agricultural Research Corporation (Embrapa – Genetics and Biotechnology Resources). “Afterwards we’re going aim for the production of a bovine transgenic gene. Since we already master this technology, our expectation is that we’ll manage to produce an animal with the human factor IX gene by 2007”, the scientist says.

The breeding of the genetically modified mice, the research’s initial starting point, was completed about one year ago. The work was coordinated by the chemist João Bosco Pesquero, vice director of the Experimental Model Developments in Medicine and Biology (Cedeme) of UNIFESP, and had the participation of professor Luiz Eugênio Araújo de Moraes Mello, the center’s director general, the biologist Heloisa Allegro Baptista, responsible for the transgenic animal laboratory, as well as the biomedical doctor Fabiana Louise Teixeira Motta. Mice possess, as well as a resemblance in the working of the human organism, a series of advantages over other animals. “The cost of maintaining mice is ten times less than that of goats or cows and the gestation period and development of the animal is a lot more rapid, which allows us to know if the experiment was or was not successful. In three months the offspring have been born, have left their mother and are ready for breeding”, says Pesquero. The confirmation that the offspring are or are not transgenic is done one month after birth. Small parts of the ear or tail are submitted to DNA analysis in order to evaluate if the exogenous genes are or are not present in their genomes.

Founded in 1996, to substitute the Central Breeding Laboratory, the Cedeme is the only national laboratory, and one of only two in South America – the other is located at the University of Buenos Aires, in Argentina – with the capacity to locally produce transgenic models of animals for other researchers. These mice, produced from the removal of any  gene (knockout model) or by way of the addition of an exogenous gene (genetic addition model) have various scientific and biotechnological applications. “In the area of medicine, we’re creating animal models for studying human illnesses, such as hemophilia, hypertension, diabetes, Alzheimer’s disease and cancer, and to develop drugs and therapies for combating them”, says Pesquero. “Ethical restrictions and cost are a lot smaller for carrying out experiments on these animals.”

UNIFESP’s genetically modified mice are less than 10 centimeters in length and are also ideal for studying the function of human genes. “With the recent sequencing of the human genome, we can verify that we don’t know the function of the majority of the genes present in out genome. To this end, the transgenic animals are important tools for obtaining information concerning the functions of these genes.”

Furthermore, the current tendency within the pharmaceutical industry is to make a “humanization of mice” in order to use these models in the development of new drugs against various illnesses. In this process a gene of the rodent is removed from the animal’s genome by the knockout technique knockout and a human gene is inserted by the process of genetic addition. This process should considerably facilitate the development of new medicines, lowering costs and shortening the time for the new drug to arrive at the pharmacies. “Another major advantage of our work for the country is technological independence. In the long term, it’ll also result in economy for researchers that need to import animal models for very high sums of money. Our mice cost around US$5,000 a pair, while for those imported the price can reach ten times that value”, advised chemist Pesquero. For him, there is a good market for these animals in the country. ‘Between 50 and 100 Brazilian scientist groups exist, carrying out research with transgenic animals and many don’t know that we have this know-how. As well as this, our models could be sold to scientists of other countries.”

The first transgenic created by the group was the mouse named Vítor, born on the 24th of December 2001 (see Pesquisa FAPESP No.75). The rodent was produced using the duplication of the B2 receptor of kinin, a substance associated with inflammation and hypertension processes. Although it was not the first genetically modified mouse developed in Brazil, the UNIFESP laboratory is the only one in the country that today is structured to provide transgenic animals by the technique of pronuclear microinjection, the most widely used by scientists in the world. “The major advantage of this method in relation to the others is its efficiency in production. The rate of positive animals, those that contain the gene inserted into its genome, is much superior to the other methods”, explains chemist Pesquero. In this technique, the fertilized egg is collected moments after copulation. Before the cellular nuclei of the spermatozoid and the egg, called pro-nuclei, hook up, a solution with copies of the exogenous DNA is injected into one of them, which can be from individuals of the same species or of another (for example, human genes). The next step is to transfer the embryo to the uterus of a female host procreator, the so-called “surrogate  mother”, where the offspring will be developed.

Another advantage of this technique is that it permits the obtaining of animals that express the exogenous gene in all of their cells, including the germination cells (egg or spermatozoid), guaranteeing transgenic progeny. “Although more efficient, the technique of pronuclear microinjection is also more expensive and demands special equipment, such as inverted light microscopes and micro-manipulators, as well as highly qualified personnel”, explains Pesquero.

After the birth of Vítor, the UNIFESP team has already managed to successfully produce other two rodents with genetic alterations, as well as that with the gene of the factor IX protein. One of these animals, a mouse (Mus musculus), received the addition of a rat gene (Rattus norvegicus), a producer of the enzyme tonine, which, it is believed, is related to hypertension processes. This animal was generated by the researcher Jorge Luiz Pesquero, João Bosco’s brother and a professor at the Federal University of Minas Gerais (UFMG) and the Mogi das Cruzes University (UMC). “In experiments, in vitro, the tonin participates in the process of the releasing of angiotensin II, a substance that produces vein constriction (reduction in the diameter of the blood vessels). To know definitely if it is truly involved in the birth and the maintenance of arterial hypertension, an alternative is to study transgenic animals that had this enzyme produced in an amplified scale”, affirmed the scientist.

Hypertension and memory
After two years of work, we’ve obtained very interesting information. Our data points in a literally different direction than that which we had expected. The animals with the exogenous genes of tonin are slightly hypertensive, but not because of the liberation of angiotensin II. Now our challenge is to discover what substances are involved in this hypertension process’, says Jorge Pesquero. The research results are important because they could help in the treatment of millions of patients with hypertension on the planet. The other transgenic mouse born at UNIFESP received a DNA sequence to produce a large quantity of a protein that could be involved in the regulation of aspects of eating behavior and of memory. This study is being carried out by the researcher Beatriz Castilho, from the same institution. “Our research is still at the beginning. We received the first transgenic animal from Cedeme some four months ago”, said the researcher.

Right from the creation of the first transgenic mouse, some four months ago, the Cedeme scientists have managed to perfect their working methodology. The proof of this is that for the creation of the first it was necessary to inoculate 500 embryos. Today the success rate is much higher, in the order of 1%. Thus for each animal produced, on average, one hundred embryos had been inoculated. The mastery of the technique has also brought into play the production of animal models. As well as the production of these rodents, Cedeme is also working with creation-preservation, a technique that consists in the storage of spermatozoids or embryos of transgenic animals to preserve their lineages (or descendants). “This is an important area because it is much cheaper to maintain this bank of germoplasma that conserve the animals alive in captivity.”

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