Knockout rats have arrived

Scientists have created a knockout rat that finally opens the model organism to the kinds of experiments that have only been possible in mice and some non-mammalian species, they report online today (August 11) in Nature.

Image: Wikimedia commons,
Janet Stephens

"We're finally going to enable genetic manipulation in the most widely studied and well characterized animal model of human disease," said molecular geneticist linkurl:Aron Geurts;http://www.mcw.edu/HMGC/Laboratories/AG.htm of the Medical College of Wisconsin, who was not involved in the research. "That's very exciting to everyone." Rats have long been a popular model system for many aspects of biomedical research, but when it came to genetic manipulation, the mouse was the system of choice. "We use the mouse because that technology was well developed and was being refined so as to allow us to make more and more sophisticated and specific alterations," said cancer biologist linkurl:Tyler Jacks;http://web.mit.edu/jacks-lab/index.html of Massachusetts Institute of Technology. Rats simply weren't an option because no one had yet figured out how to culture their embryonic stem cells (ESCs). Last year, with a few tweaks to the culture media, stem cell biologist linkurl:Qilong Ying;http://www.usc.edu/programs/pibbs/site/faculty/ying_q.htm and his colleagues at University of Southern California finally succeeded in culturing rat ESCs, making it possible for them to use a technique often used to create knockout mice -- homologous recombination in ESCs. Swapping out some DNA in a region of the rat genome to disrupt a particular gene, the tumor suppressor gene p53, the team then injected the altered ESCs into developing embryos to create chimeric rats, which contain both manipulated tissues (derived from the injected ESCs) and unmanipulated tissues (derived from the cells already present in the rat embryos). When the rats' germline cells (sperm and eggs) happen to be derived from the manipulated cells, those rats can pass those genetic changes onto their offspring, and thus be bred to create complete knockout rats. There were some minor "technical issues that we needed to overcome before we could make the knockout," Ying admitted, but the real hurdle was culturing the rat ESCs. Once they figured out how to do that, the generation of knockout rats via homologous recombination was relatively simple. Now "you can target any gene you want, delete just one amino acid or whole genes, [and] use an inducible system [to target a] gene to be expressed at a certain time in certain tissues," he said. "The investigator has full control over the type of mutation they want to make." It's not the first time scientists have made a knockout rat, however. Another technology, employing protein structures known as zinc finger nucleases (ZFN) to target genes in single-celled rat embryos, has already been used by Geurts and others to target and disable specific genes in the rat genome. While this technology is still useful, Geurts said, the homologous recombination technique "will allow us to do many types of experiments that you cannot do with ZFN." Specifically, the ESC technology provides greater control when creating genetic knockouts, and could also make it easier to generate knock-in rats as well, in which new genetic sequences are added to the genome, Geurts said. This technique is also cheaper than the zinc finger technology, he added, which involves costly reagents as well as the expense of raising and maintaining rat colonies. The ESC technology allows the genetic manipulations to be performed in culture before any animals are ever involved in the process, and could thus allow scientists to create knockout rat models "in a high throughput way," Geurts said, like is being done in mice to "knock out every gene in the genome." Furthermore, this technique is likely to be more widely adopted because it is similar to the techniques scientists have been using in mice for years, added molecular geneticist linkurl:Ronald DePinho;http://www.hms.harvard.edu/dms/bbs/fac/depinho.html of Harvard Medical School, who did not participate in the research. "This particular technology is more aligned with the couple of decades of experience that we now have in manipulating the mouse genome," he said. "[Thus, it] would dovetail very well with the existing body of reagents designed to manipulate specific genetic sequences." And in general, it's always helpful to have additional model species to analyze gene function on the organismal level, DePinho added. "Yeast, C. elegans, Drosophila, the mouse, zebrafish -- these have all had significant impact on biomedical research," he said. "To have the rat join that pantheon of important model systems on the level of being able to manipulate its genome is a very important advance for all of us." C. Tong, et al., "Production of p53 gene knockout rats by homologous recombination in embryonic stem cells," Nature, AOP, doi:10.1038/nature09368, 2010.
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