Friday, April 27th, 2002, Panorama farm, Campinas. In the delivery unit, everyone at their posts and great expectations. Something more than a caesarian birth was under way: this was the first cloned heifer to be brought into the world using an adult cell from a Nelore cow. Responsible for the project, the team led by José Antônio Visintin, from the Faculty of Veterinary Medicine and Zootechny of the University of São Paulo (USP), was waiting impatiently. Finally, it was born: except that, instead of the expected heifer, it was Marcolino, a strong and healthy male.
Everyone was delighted with the success and astonished by the surprise. “It is impossible to generate a male clone from an adult female cell, as the sex is already defined in the somatic cell”, is the explanation given by Visintin, who coordinates two research projects of cloning cattle: one from an adult cell and the other from a cell from a fetus. The team worked hard for two years, taking turns, over 24 hours a day, to check cells and the stages of the cloning process, every two hours.
With this effort and the data under control, Visintin soon found out the reason for it all: “I checked all the procedures used over these two years, and I discovered that on June 20th two groups of cells were frozen: one of adult cells from a female, and the other of cells from a fetus. In spite of the all the precautions, someone probably more affected by the exhausting routine of the process wrapped up cells of a fetus, thinking they were adult cells”. This is part of life, science showing its reality. As the team has been doing fertilization in vitro for six years, without any problems before, it could only have been an accidental swap. But it had to be proved.
Having ruled out the possibility of the cow having been covered by a bull, an examination of DNA was carried out on samples from the Nelore donor, the receiving cow, the newly-born, the culture of the cells from the donor and from the culture of the cells from the fetus. With the support of the team of José Fernando Garcia, from the São Paulo State University (Unesp) in Araçatuba, which is taking part in the Genome Project, and of the examinations carried out by the LinkGen laboratory, the genetic mapping confirmed it: Marcolino is a clone that came from the cell from the fetus, which confirms the involuntary swap.
“Our idea was to develop first the clone from the adult cell: we would have until the end of 2002 to do this. Only afterwards would we set off for cloning from the somatic cell of a fetus”, Visintin reveals. The mistake therefore turned the research projects around and anticipated the objectives. “The most important thing”, the researcher stresses, “is that we did the first cloning from a somatic cell in Brazil and proved that we have the technology to produce it. A different result from the one reached by Embrapa in Brasilia, which produced a female cloned from an embryonic cell”.
For him, the objective of cloning is to reproduce animals in extinction or for high production, reproducing more productive dairy cows, and for transgenics that produce high quantities of components of pharmacological interest – the case of coagulation factors 8 and 9, very important in treating hemophiliacs, and which can be produced in milk. “Given that cows don’t have Aids, these factors could be taken out and supplied to humans, without the risks of blood transfusions”.
The first step for the team was to collect and to cultivate cells from the ear of an adult Nelore cow. In parallel, a culture was made with cells from a fetus of an undefined race, between 45 to 60 days of gestation and 20 to 30 centimeters in length, gathered from abattoirs, removed from the entrails from slaughtered cows. The fetuses were put into an appropriate solution and container for preserving the cells and taken to the laboratory of the College of Veterinary Science, at most 3 hours after the death of the cow. The fibroblasts obtained from the two cultures were frozen separately.
In parallel, ovaries were taken from cows slaughtered at the abattoir and their oocytes (ovules), aspirated in the laboratory. Of those that had already matured, the nuclei were removed, obtaining enucleated cells (without nuclear DNA). A fibroblast from a culture, deprived of the bovine fetal serum for two days, at the G zero stage (the beginning of the development of the cell), was next inserted into the enucleated ovule. An electric shock was applied, which unleashed a cellular reprogramming of the cytoplasm: the cells began to split up. After seven or eight days in culture had gone by, the blastocyst stage was reached, with the start of the formation of the cells of the placenta and the embryonic button. Then, the future clone was transferred to the uterus of the surrogate cow, carrying on with the nine month gestation.
Losses occur at all the stages of this process. Of every 100 oocytes taken out, for example, only some 80% mature. When taking the nucleus out, there is a new loss of about 50%, leaving some 40 enucleated oocytes. After the insertion of the fibroblast and the electric shock, only half of the oocytes merge, which allows 20 of them to be cultivated. Of these, finally, only some five develop and reach the stage of a blastocyte, to be implanted in the cows.
After gestation has started, there are also losses. The most critical periods – with a great incidence of being badly fixed and of abortions – are the first 20 days of gestation, the following stage of between 20 and 50 days, and at 90 days. And sometimes the calf dies at birth, from malformation of its organs. “Marcolino is the first success in two years of research”, says Visintin. “Out of 16 blastocyte implants, we achieved four gestations. ne of them resulted in an abortion, and we have another two cows pregnant with clones produced by my team: one at six months of gestation and the other at two”.
He recalls that the level of losses leads to important reflections, since research with human beings always results from animals”. “If today human fertilization in vitro is well developed, it is because this process has been commonplace in animals for many years”. With an essential difference. “When cloning cattle, which is still in the early stages, if something goes wrong, the animal can be sacrificed “.
With the clones obtained from adult and fetal cells, the group is going to compare the development of both to answer certain questions: “Is the clone originated from an adult cell old at birth? If I make two or three from one cell, do these cloned cells obtained at each repeat grow old too?” To find out, a clone from an adult cell has to be obtained as soon as possible. “We will make the clones available for studies in physiology (biochemical and sexual) and genetics. The team led by Maria Angélica Miglino, from the Anatomy Department of the Faculty of Veterinary Medicine, will analyze the placentas from each gestation, to check the form, composition and function, compared with placentas from normal gestations, which should contribute towards the discovery of the reasons for failures.
To guarantee that there will be no new accident, the researcher from USP has taken steps to separate the laboratories completely: one only for cloning with adult cells, and another for fetal cells. With the premature success of the second stage, he hopes that the articles about Marcolino and a molecular study of him may be ready for publication by the end of the year.
1. Maturation and Fertilization of Oocytes and Co-Cultivation of Bovine Zygotes in a Primary Culture of Epitelian Cells from the Oviducts and Permanent Lineages (nº 96/10634-0); Modality Regular research benefit line; Coordinator José Antônio Visintin – University of São Paulo; Investment R$ 127,290.00
2. Cloning of Bovine Embryos: Development of the Technique for Nuclear Transfer; Modality Regular research benefit line; Coordinator José Antônio Visintin – University of São Paulo; Investment R$ 312,716.01