Ever since the first rats were brought into laboratories nearly 200 years ago, they have been a mainstay of medical research. Inbreeding the animals has led to hundreds of strains with health defects that resemble those of humans, providing models that can be used to study disease, the effects of drugs, behavior, and other aspects of health. But in the age of modern genetics, research into the rat has been limited by a lack of tools to manipulate its genes, meaning that this important model has lagged behind other animals such as the mouse and fruit fly. That's unfortunate because rats are more closely related to humans and can potentially reveal more about our diseases. Two recent studies by the group of Zsuzsanna Izsvák and Zoltán Ivics, published in the journals Nature Methods and Methods, are now giving researchers the tools to help the rat – and likely other model organisms – catch up.
The first step in genetic engineering is to construct a new bit of DNA that will be inserted into an animal in order to add a gene or interfere with an existing one. In the past, the change has often been made in an embryonic stem cell, grown and transformed in the lab and then implanted into an animal embryo at a very early stage. This process has only recently been mastered in the rat. It is complicated and sometimes results in "patchwork" animals; some of their cells make use of the new DNA and others don't. And researchers have to wait until animals are born to discover how the gene behaves in specific tissues.
A more efficient procedure is to add the new information to an animal's germline stem cells, which eventually develop into sperm or eggs. Such cells can go on to produce thousands or millions of new cells, each of which bears the altered DNA. When they fuse with another reproductive cell, the new embryo has it as well. This method is already widely used in mice, and Zsuzsanna and her colleagues have now adapted it successfully for the rat. Scientists already knew how to grow their germline stem cells in the laboratory and then reimplant them in animals. But a significant barrier was a lack of methods to alter the cells' genetic information in the first place, and the new studies propose a solution.
Over the past decade, Zsuzsanna and Zoltán have turned to molecules that act as "natural" genetic engineers to overcome this problem. Transposons – also known as "jumping genes" – are bits of DNA that naturally copy themselves and jump around to new positions on chromosomes as cells divide. They arose long ago in ancient organisms, possibly through infections by viruses that inserted genes into cells. Gradually the transposons spread to create millions of copies that have left their footprints throughout the genomes of humans and other organisms.
Sometimes when such a gene jumps, it lands in the middle of another gene and disrupts its function. This makes transposons dangerous, and over hundreds of years of evolution, most of them have acquired mutations that have defused them by removing their ability to copy themselves and jump.
But these features caught the interest of Zsuzsanna and Zoltán, who thought that transposon behavior might be tweaked and used to disrupt an animal's genes or insert new ones. The first step, which required years of work, was to reactivate a transposon – using sophisticated computer programs that detected mutations and could recover the original gene sequence. The labs first achieved this with a transposon called Sleeping Beauty. Once they had an active form of the molecule, they introduced new mutations that made it jump at a higher rate, rendering it active enough for use in genetic engineering. In the meantime they have rebuilt other transposons in a similar way.
The new tools have already been used to manipulate the genes of mice and other animals. But the rat has been high on the researchers' minds due to the lack of methods to manipulate its genes. In the latest studies, the researchers introduced the transposon into cultures of germline cells. Since only a proportion of the cells insert the transposons into their genomes, the scientists added an antobiotic resistance marker to detect cases in which it had been successful. They isolated these cells and allowed them to divide to create millions of identical copies, which were then implanted in male rats. The animals went on to mate and produce offspring with the altered genes.
In the end, the scientists had 35 new strains of rats with specific genetic mutations, about half of which involve genes that have been linked to disease. After years of work, it's a promising payoff. Soon, Zsuzsanna and Zoltán hope, the method will become routine, allowing researchers to learn much more from many existing strains of rats – and to develop new ones – that mimic human diseases. And the approach is not limited to rats; it can likely be adapted to many other model species for which scientists do not yet have an effective toolkit for genetic engineering.
- Russ Hodge
Generating knockout rats by transposon mutagenesis in spermatogonial stem cells. Izsvák Z, Fröhlich J, Grabundzija I, Shirley JR, Powell HM, Chapman KM, Ivics Z, Hamra FK. Nat Methods. 2010 Jun;7(6):443-5.
Sleeping Beauty Transposon Mutagenesis of the Rat Genome in Spermatogonial Stem Cells. Ivics Z, Izsvák Z, Chapman KM, Hamra FK. Methods. 2010 Dec 2