Using homologous recombination, scientists from the Keck School of Medicine at University of Southern California (USC) have created the first knockout rats from modified embryonic stem (ES) cells. The new technique promises to provide models for human disease and gene function studies.
Mice have been a popular model in disease research because of the relative ease with which knockout mice have been created, but the rat is considered by the research community as a better model for the study of a wide variety of human diseases including cancer, diabetes, multiple sclerosis. Compared to mice, rats are more physiologically synchronized with humans, and their larger size makes medical procedures, such as tissue sampling and nerve recording, easier to perform. (See our April 2010 Tech News feature for a deeper investigation of the differences.)
Although homologous recombination is a well-known technique for targeted mutations in ES cells, researchers have struggled for almost 30 years to develop authentic rat ES cells to create knockout rats. To create knockout rats, researchers had no choice but to use other, less precise, techniques like zinc finger nuclease (ZFN). According to team leader Qilong Ying, assistant professor of cell and neurobiology, ZFN has a higher possibility of error than homologous recombination, leading to incomplete knockout of the target gene or inadvertent knockout of an untargeted gene. “But using the homologous combination, everything is predetermined,” said Ying.
In 2008, Ying’s team discovered that the key to maintaining ES cells is to block cells from differentiating. They developed a cocktail culture medium—called 2i/3i which contains either two (2i) or three (3i) genetic inhibitors—that allowed them to derive ES cells from multiple mice and sustain them in culture. After uncovering this crucial step to homologous recombination in mice, the team had all the tools for attempting to grow modified ES cells in rats. Unfortunately, so did everyone else who read the paper.
“That was our concern,” Ying told BioTechniques. “That’s why we tried very hard to be the first ones. We knew that other groups—in the United Kingdom, United States, Japan, and China—were doing similar work, trying to make the first gene knockout rat using ES cells.”
Two years later, they again beat the rest in creating authentic ES cells from rat blastocysts. The researchers knocked out the tumor suppressor gene P53 using homologous recombination to transmit a mutation of the gene through the germ cell line of ES cell rat chimeras.
“We believe that many labs around the world will take advantage of the well-established ES cell–based gene targeting technology to produce new rat models,” said Ying.
Going forward, Ying’s team plans to create knockout rats lacking a variety of different genes. Of special interest to Ying are immune-deficient knockout rats for transplantation applications, to overcome immune rejection of implanted cells from another species. “Our eventual goal is to derive ES cells from a pig,” said Ying. Because pig organs are similar in size to human organs, there is a possibility that pigs can grow human organs for transplantation operations by inserting human genes into pig ES cells.
But before this can be accomplished, the researchers need to derive true ES cells from pigs. So far, the researcher’s attempts have been unsuccessful. Ying believes that the 2i/3i culture medium may only be suitable for rodents, so more research for deriving these ES cells from larger animals will be necessary. But the end goal justifies the hard work, for Ying. “I think this is going to save a lot of people’s lives.”
The paper, "Production of p53 gene knockout rats by homologous recombination in ES cells," was published in 9 September 2010 issue of Nature.