by Caitlin Smith
Knocking out a gene from an animal’s genetic blueprint is a powerful tool—one that has certainly seen improvements since its relatively recent inception. Indeed, the method of removing (or otherwise taking out of commission) a gene from an organism’s DNA complement is a potent technique that is still being refined, as well as broadened to include an ever-growing list of species. Here are a few of the latest developments in the technology of tools for generating knockouts.
New media net new knockouts
For decades, molecular and cell biologists have relied on knockout mouse technology to analyze the functions of genes within an organismal context. For example, researchers use knockout mouse technology to create murine models of human diseases, so that more invasive therapies can be studied in non-human model systems. However, comparing mouse pharmacological data with rat behavioral data is not as ideal as using the same species for the entire study. Furthermore, some of the diseases under study, such as neurological ones, also include behavioral aspects that are more readily studied in rats than in mice.
Research from Qilong Ying’s lab of the Stem Cell Center at the University of Southern California recently developed a media to help create knockout rats. “The technology that we utilized to generate the knockout rat is the conventional gene targeting via homologous recombination,” says Chang Tong, a postdoctoral research fellow in Ying's lab. “This method has been used for generating gene knock out/in mice for over 20 years by transgenic facilities all around the world. It has been proved to be reliable and easy to handle.” Ying’s lab developed a “2i medium” (named for the ERK and GSK3beta inhibitors) for ES (embryonic stem) cell culture that allowed them to create real ES cell lines from rats for the first time. “The availability of authentic rat ES cell lines makes it possible for us to generate gene knock out/in rats using conventional gene targeting methods,” he says.
Tong notes that the biggest challenge in generating knockouts is how to derive ES cell lines. “Currently, scientists all around the world, including us, are trying to dissect the mechanism of ES cell self-renewal,” says Tong. “Once this question is addressed, we can develop the medium to derive the ES cell line from any other species. A universal medium that can derive authentic ES cell lines from all mammalian species is just what we want to get but does not exist yet.
In the future, I hope that we can generate gene-targeted big animals such as pig, which could be a great donor for organ transplantation in clinical application.”
Targeted knockouts and knock-ins
Recently, Sigma Life Science introduced their CompoZR™ Zinc Finger Nuclease technology as a tool for making targeted knockouts in cell lines. “Creation of knockout cells was once a very inefficient if not impossible process,” says Supriya Shivakumar, global commercial marketing manager for functional genomics at Sigma Life Science. “With ZFN technology, it is now a matter of carrying out a simple transfection.” Furthermore, Sigma’s ZFN technology can be used to create knockout animals—animals of any species or substrain. “For the past quarter of a century, mouse has been the only mammal for which knockouts could be created,” says Shivakumar. “Our ZFN technology lifts this limitation.”
Zinc fingers work by recognizing a three base pair codon in a nucleotide sequence. “Multiple zinc fingers can be combined to recognize very specific regions of sequence, in essence targeting a specific gene or target sequence in the genome,” says Phil Simmons, marketing and business development manager for Sigma advanced genetic engineering (SAGE) labs at Sigma Life Science. “Once a break has been made using a zinc finger nuclease, the cell repair mechanisms can repair the break correctly—or in a proportion of cases, incorrectly—creating insertions and deletions that result in inactivation of the gene. In addition, a sequence of donor DNA can be introduced into the system and integrated into the cut sequence. This process, called knock-in, allows for researchers to introduce a desired sequence or gene into the organism, which is then permanently part of that organism’s genome.”
Sigma’s SAGE labs use ZFN technology to create new animal models of human diseases. They can make a knockout animal in about five months—work that may have taken more than a year using classic ES cell technology. “[Creating knock-ins] will lead to even more predictive animal models of human disease,” says Shivakumar. “For example, you can now imagine replacing the rat metabolism pathways with human equivalents, thereby creating a much more relevant and predictive model for the testing of drugs.” ZFN technology works in human stem cells, creating many options for future work. Additionally, it can knock out multiple alleles; many of the cell lines used in the lab today have skewed karyotypes and often contain multiple copies of chromosomes. “We have used the ZFN technology to create a cell line that had a knockout of all four of the alleles of Bax that were present in that cell line—all simultaneously in a single ZFN experiment,” says Simmons.
Managing and preserving knockouts
With the ever-increasing number of knockout and knock-in animals and cell lines being created, The Jackson Laboratory has dedicated its efforts to systems of management and preservation of the new strains. “Our best examples of these are systems to reduce genetic drift [Genetic Stability Program (patented)]; our high-throughput application of in vitro fertilization and embryo cryopreservation; and more recently, our highly efficient approaches to the cryopreservation of mouse sperm,” says Michael Wiles, senior director of technology evaluation and development at The Jackson Laboratory. “This last method allows strains to be economically frozen down, whilst allowing their very rapid and scalable return to ‘the shelf’ upon demand. The NIH Knockout Mouse Project and its international partners are generating public resources of mouse embryonic stem (ES) cells containing a targeted null mutation (many conditional) in every gene in the mouse genome. It is expected that many of these ES lines will be ‘turned into mice’ in the next 3-5 years, heralding in a new era in our understanding of gene function. Thus with potentially 10,000 new mouse strains coming on line in the next ten years, cryo-mouse management systems will be essential to control costs and provide effective distribution of strains.” The Jackson Laboratory is also testing new ways to improve ES cell germ line transmission efficiency, to allow faster—yet more controllable—flow of modified ES cell lines to germline.
Wiles emphasizes the importance of mouse management systems at the The Jackson Laboratory, a potentially very expensive endeavor. “With the creation of all these strains (at great expense) a major up-and-coming problem will be their management (potentially, an even greater expense),” he says. “This will be further exasperated by the development of knockout strains on multiple genetic backgrounds.”
Another exciting use of knockout technology relates to human tissue grafting. “This is being done by inclusion of human genes and/or the genetic modification of animals to receive and support human tissue (xenograft models), which can then be used in a research maintaining context of the complete organism,” says Wiles. “Human cells can be engrafted into these strains. These mouse strains can then support and provide a research environment for aspects of human hematopoiesis, islet transplantation, and cancer stem cell development.”
While our ability to create genetic models has made more progress than anyone could have imagined, “our ability to understand how a gene interacts within a whole organism, and the way that organism can adjust to its removal or mutation, is still very primitive,” says Wiles, noting that perhaps bioinformatics will be key to understanding this problem. “We have quantum leaps in data, now we need quantum leaps in knowledge.”