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Guinea pigs made to measure

Unifesp team consolidates the pioneering production of transgenic miceobtained through the technique of pronuclear microinjection

Every morning a researcher opens the door, always kept locked, of a Federal University of São Paulo (Unifesp) laboratory and alters the order in which the thirty cages containing white, black and grey mice (Mus musculus) are laid out. So, only the house team can identify, by the code of each cage, the first transgenic mice – genetically modified -, produced there through a technique unprecedented in Brazil. It is hoped that the technique, already used worldwide in the study of illnesses such as Alzheimer’s disease, diabetes, hypertension and cancer among others, can be applied to animals of larger size for the production of vaccines and medicines.

João Bosco Pesquero, the laboratory coordinator of transgenic animals at the Center for Development of Experimental Models for Medicine and Biology (Cedeme) of Unifesp, adopted the change of routine after the birth of Victor on the 24th of December of last year. Victor is not the first genetically altered mouse bred in Brazil – a group from the University of São Paulo (USP) obtained the first in July of last year -, but he represents the domination of yet another technique: pronuclear microinjection.

By this method, the fertilized egg is collected a few hours after copulation. Before the pronuclear cells – nuclear cells of the spermatozoid and the egg – hook up, a solution with copies of the desired genes is injected into one of them – of individuals of the same species or of another. Then the embryo is transferred to the uterus of a “surrogate mother”.

This technique, the most widely adopted in the world for the addition of genes, allows one to obtain animals that express the desired gene in all of the cells, including the germinative cells (the egg and the spermatozoid), which guarantees that the descendants will have the genetic change. Already the transgenics at USP have been bred through the manipulation of the stem cells, which maintain the capacity of differentiating themselves into any type of tissue. These animals, which have some cells genetically altered and others with an integral genome, are called chimerical animals. What is important is that the germinative cells possess the change, since only then can the animals pass on the desired gene to its offspring and they will be considered transgenics.

As well as greater efficiency in the generation of transgenics by inheritance acceptance, another advantage of microinjection is the capacity for its use with other laboratory species – rats, rabbits, cats and dogs -, as the manipulation of stem cells is limited to mice. Nevertheless, both methods do not allow for the control of the number of copies of the desired gene that will be incorporated by the genome nor the region of the chromosomes to which they will attached – consequently, there is always the risk that the intrusive sequences block essential genes or even cause death.

Differences apart, transgenic animals are considered to be essential for the study of the disease development and the action of genes whose function genes is ignored. Indeed, Victor’s heart cells and those of his first descendants have already produced a protein, the receptor of bradykinin B-2, in a quantity higher than normal. This molecule links itself to bradykinin, a peptide liberated by the cells during the inflammatory process and which promotes the dilation of the veins.

Mass production
In this way Brazil is beginning to overcome twenty years of delay, since the appreciation of the theme is only recent here. A researcher who spent four years working on the development of transgenic animals during his post doctorate – at the University of Heidelberg and at the Max-Delbrück Institute for Molecular Medicine in Berlin, both in Germany -, João only managed to begin research at Unifesp in 1997.

To obtain Victor, the team had to inoculate five hundred embryos, transferred to the uterus of thirteen females. Afterwards the results improved: with another one hundred and eighty embryos, the laboratory also produced two genetically modified females, born at the end of February. The research forecasts that in May, when the laboratory will be working at full capacity, two hundred embryos will be inoculated per day. This work should spawn from two to four transgenic mice, since the efficiency of the method lies between 1% and 2%. Monthly production, estimated in forty to eighty animals, could even surprise other institutions. “We are going to turn ourselves into a transgenic factory” says the researcher.

Right away the production should save time and money. Currently, to import a genetically modified animal there is a delay of from six months to a year, depending on the lineage requested, and demands patience to confront bureaucracy and the customs. Normally a transgenic mouse couple (male and female) costs around US$ 2,000 but the price can even increase fourfold, according to the importance of the animal. At Unifesp, it is calculated that the cost of a couple will come out at around US$ 1,000.

Two research groups have already shown an interest in Unifesp’s mice. Jorge Luiz Pesquero, João Bosco’s brother, coordinates one of them at the Biological Sciences Institute of the Federal University of Minas Gerais (UFMG). In partnership with Unifesp, Jorge intends to study the role of the enzyme tonin in the origin and in the maintenance of arterial hypertension. It is already known that tonin participates in a reaction that transforms angiotensinogen I into angiotensin II, a substance that produces vasoconstriction (decrease in the width of the blood vessels).

The two teams, which since 1997 have been attempting to obtain mice that produce tonin in a higher quantity in several tissues, were successful in February of this year. The two female mice born at Unifesp have copies of the gene that produces tonin in the astrocytes, cells of the nervous system with abundance of angiotensinogen. Because of the genetic alteration, these cells begin to overproduce the enzyme. With the animals of this lineage, Jorge intends to investigate not only hypertension, but as well the capacity of memory and of learning – there is evidence of a greater quantity of tonin in the areas of the brain is linked to these functions.

Another team, from Embrapa (Brazilian Agricultural Research Company) in Brasilia and from the State University of Campinas (Unicamp), want to make use of mice for the creation of biofactories – animals that produce biological molecules. Transgenics of large size (bovines or goats) would produce proteins in their very own milk of nutritional or pharmacological interest. However, before inserting into these animals the genes that codify the desired molecules, one needs to carry out tests on mice, which have a short reproductive life cycle: gestation takes only twenty days, while of a cow lasts nine months. “This quickness allows us to analyze the incorporation of the genes within a few months, and, if necessary, to redirect the research”, says Elibio Rech, the transgenics program coordinator at Embrapa.

Over the next few months, in collaboration with Unifesp, the Embrapa group will attempt to obtain transgenic mice that express the gene scFv (single chain fragment variable). This gene produces an antibody against breast cancer, which could be used in diagnostic exams and even as a vaccine against the tumor. The researchers don’t only want to work with mice. The plan is to employ the pronuclear microinjection technique to obtain rats (Rattus norvergicus), larger animals of between eighteen and twenty five centimeters in size. As well as making the work easier – the manipulation of the small mice, which don’t reach ten centimeters in length, has to be done using a magnifying glass – the rats are important because a large part of what we know of the working of the human organism has been based on research done with them.

The Project
Setting up of the Laboratories for the Production and Maintenance of Transgenic Animals (nº 99/05457-0); Modality Multi-use equipment;
Coordinator João Bosco Pesquero – Unifesp; Investment R$ 177,209.72 and US$ 166,389.44