A room a little bigger than 11 meters square in the new wing of the School of Veterinary Medicine and Zootechny of the University of São Paulo (USP), with a door that is always closed to strangers, houses a little great feat of genetic engineering. In this cramped space, where two air-conditioners and an exhauster guarantee a temperature controlled at between 22 and 25 degrees Celsius and lamps simulate natural light for 14 of the 24 hours of the day, there is a biotron at work with special guinea pigs. Inside the plastic cages arranged on the shelves that take up the two largest walls in the room, lives the first batch of genetically modified mice in Brazil. These animals, over 30 of them – the first was born in July, but the number may vary from week to week, because of further mating and possible casualties – bear alterations in the Fbn1 gene that is responsible for the synthesis of fibrilin 1, an essential protein for the formation of the conjunctive tissue.
A portion of these rodents has received an inert version of Fbn1, which simply does not produce fibriline. Another received a copy of Fbn1 that gives rise to a degenerated form of this protein. In human beings, this second genetic mutation leads to Marfan’s syndrome, a rare hereditary disease that causes excessive growth of limbs, dislocation of the retina, and cardiovascular problems, limiting life expectation to 40 years. The two lineages of the GM mice were obtained through the manipulation of the stem cells of the embryos, the most advanced methodology available nowadays for this purpose.
The GM rodents, made in Brazil, therefore represent Brazil’s independence in an important field: the production of made to measure guinea pigs for the study of health problems of a genetic origin. “Mastering this technique, we will be producing our own model animals for studying a series of diseases that affect mankind”, says Lygia da Veiga Pereira, the coordinator of the Molecular Genetics Laboratory of the Institute of Biosciences at USP, which carried out the manipulation of the rodents’ stem cells. “It will be more practical and we will save money as well.”
The biotron with the guinea pigs is housed by Veterinary Medicine, because there was no physical space in Lygia’s laboratory to accommodate it. In addition, the veterinary researchers, under the coordination of José Antonio Visintin, were interested in collaborating with the IB’s experiment, by setting up a biotron for the mice, and they were interested in learning the technique, in order to employ it in the generation of large sized GM animals. “We want to make genetic alterations to pigs and cattle”, comments Visintin.
The success in the production of genetically modified mice led the researcher to receive the first order of customized guinea pigs, made by the São Paulo Heart Institute (Incor) by the Oswaldo Cruz Foundation (Fiocruz) of Rio de Janeiro. At the moment, Brazilians who need GM guinea pigs need to import this kind of animal. The cost of these modified rodents may be low, if they belong to lineages developed abroad. Or very high, in the region of thousands of dollars, when it is a question of an animal with a genetic alteration not yet carried out by any laboratory in the world.
Obtaining genetically modified animals depends on a full mastery of the techniques for manipulation and culture of the stem cells of embryos, one of the areas of research that most sparks off debates these days, because these cells can be used for such purposes as the cloning of species. Dealing with this kind of cell arouses the most varied polemics, above all if they are from human embryos. Why? Because the embryos from which the stem cells are extracted end up dying, after this material is removed. As she does not work with stem cells of human embryos, Lygia does not face this ethical dilemma.
In practice, her first challenge in the production of GM mice was to get hold of good stem cells of the rodents’ embryos, or, in the language of molecular biologists, highly pluri- or totipotent stem cells. What exactly are these cells and what are they used for? They are the primordial, undifferentiated cells that originate all the cells of an organism with specific functions: nerve, blood, muscle, germinative, cardiac and the other cells. Accordingly, the introduction of a genetic modification in the DNA (deoxyribonucleic acid, which carries the genetic code and is present in all cells) of the stem cells of embryos, such as the one carried out by the IB’s molecular biologist on mice, has a great chance of being incorporated by all the kinds of cells that are going to form an organism, thus creating a GM being.
Like embryos, human beings also have stem cells. The problem is that, so far, there is only concrete evidence of pluripotency in the material taken from embryos – and not in animals of an advanced age. That is why those who dedicate themselves to produce GM guinea pigs find themselves obliged to arrange and cultivate in the laboratory the stem cells of rodent embryos, where the altered DNA is to be injected. That is precisely what Lygia did. From the blastocyst (a kind of cell that precedes the formation of the zygote-ovum) of agouti-furred mice, the biologist took out the so-called embryonic bud, from which she established in vitro strains of embryonic stem cells. Then, in the DNA of these stem cells she replaced one of the normal copies of the Fbn1 gene by one of the two versions of the modified gene, the Fbn1 incapable of producing fibriline, or the Fbn1 programmed to codify a debased form of this protein.
These modified stem cells were then cultivated and added in laboratories to morulas – cells that represent the initial stage of embryonary development, taken from fertilized ovules – from normal, white-furred rodents. This new set of cells formed an embryo that, in turn, was transplanted to a “surrogate mother”, a mouse of the female sex – and with white fur. She has the task of creating a litter of chimerical animals. Why chimerical? These animals are formed by two distinct kinds of genetically distinct cells, one coming from the original embryo, and the other from the genetically altered stem cells.
“The level of being chimerical can be estimated from the coloring of the skin, ranging from 0 to 100%”, says the Russian Alexandre Kerkis, a visiting professor at the IB, who assisted Lygia in the development of the GM mice. As the agouti tonality is dominant over the white – and the altered gene was inserted into stem cells from the agouti animals -, the darker chimeras show a greater quantity of cells derived from the modified stem cells. That is to say, it is in them that the genetic alteration has been fully incorporated into the DNA. With the lighter chimeras, then, obviously, the degree of the expression of the genetic alteration is lower.
This is the reason why, when the time comes to carry out the final matings that will result in the creation of a stable lineage of transgenic rodents, the researchers use fundamentally the agouti-furred chimeras. “The process of generating a new lineage of transgenic mice takes about one year”, comments Lygia, who learned the technique for manipulating embryonic stem cells at the beginning of the 90s, during her studies for a doctorate at the Mount Sinai Hospital in New York.
It is a lengthy process, and besides the difficulties with stem cells and genetic manipulation, there is always the risk of losing a litter of transgenic mice. In September last year, for example, a slight slip in the asepsis of the biotron made a colony of mice that was being manipulated for an experiment get mange and be discarded. A small accident that delayed the timetable of the researchers from the IB and the Veterinary School, but not one that took them from the charted path.
The future of stem cells
The animal cells that are capable of splitting indefinitely in a culture and originating others to carry out specific tasks – they form the nerve, muscle or heart tissue, for example -, are all called stem cells. There are two major categories: those derived from embryos and those that come from adults.The embryonary stem cells are more researched than the adult ones, for being more versatile. They usually come from the fetal tissue of an interrupted pregnancy or, more often, from the ovules fecundated in vitro not used by couples with problems of infertility. The ovules are stored in clinics and hospitals.
According to the stage of their evolution, these ovules may offer totipotent stem cells (that can generate all the tissues of a being, as well as the being itself), pluripotent cells (that originate the greater part of the tissues), or multipotent one (that are transformed into some kinds of cells). With the removal of the cells, the embryo ceases to be viable, a fact that gives fuel to the polemics over research with embryonary stem cells. “The question is not put properly”, in the opinion of Marco Antonio Zago, who teaches at USP’s School of Medicine in Ribeirão Preto. “These embryos have been discarded and were never going to generate a being.”
In adults, the stem cells come from the bone marrow, the umbilical cord and, to a lesser extent, from the bloodstream itself. The problem is that there isn’t undeniable evidence of pluripotency in the stem cells obtained from developed beings. “There have been some advances in this area over recent years, but the subject is still controversial”, says Zago, who is researching blood from the umbilical cord as a source of cells for diseases like leukemia.
Development of an Animal Model for the Marfan Syndrome, through the Manipulation of the Mouse Genome (nº 96/09031-9); Modality Young Researcher Program; Coordinator Lygia da Veiga Pereira – IB/USP; Investment R$ 70,382.31 and US$ 100,645.00