In Chapter 7 of our March 2010 book-length report, Animal Models for Therapeutic Strategies (published by Cambridge Healthtech Institute), we discussed recently-developed methods for producing knockout rats. These methods included zinc-finger nuclease (ZFN) genome editing and transposon mutagenesis in cultured spermatogonial stem cells. Our most extensive discussion was of the ZFN editing technology, which was developed by Sangamo BioSciences (Richmond, CA), and is the basis of the knockout rat models marketed by Sigma-Aldrich Advanced Genetic Engineering (SAGE). We also mentioned the SAGE knockout rat platform in an earlier blog post.
In Chapter 7 of our report, we also mentioned that it would now also be possible to construct knockout rats “the good old way”–using the same homologous recombination technology that researchers use to create knockout mice. Drs. Mario R. Capecchi, Martin J. Evans and Oliver Smithies were awarded the Nobel Prize in Physiology or Medicine for 2007 for having developed this technology in the late 1980s. To construct knockout mice, researchers isolate and culture mouse embryonic stem (ES) cells. These are derived from the inner cell masses of preimplantation mouse blastocyst embryos, and grown under particular culture conditions. These cells are subjected to homologous recombination with a vector containing a truncated version of the gene to be targeted, to eventually yield knockout mouse strains.
It has not been possible to develop knockout rats because the conditions for culturing ES cells worked only for a few inbred mouse strains, and not at all for either most mouse strains or for the rat. Conditions for culturing mouse ES cells are complex. They involve the use of feeder fibroblasts and/or the cytokine leukemia inhibitory factor (LIF), together with selected batches of fetal calf serum or bone morphogenetic protein (BMP). These culture conditions had been determined empirically.
In 2008, Dr. Austin Smith (Director of the Wellcome Trust Centre for Stem Cell Research, University of Cambridge [Cambridge, UK]) and his colleagues developed culture conditions that allowed them to culture rat ES cells that were capable of transmitting their genomes to offspring. These ES cells could also be used to produce knockout rats.
Dr. Smith and his colleagues realized that the standard conditions for culturing mouse ES cells expose the cells to inductive stimuli (e.g., fibroblast growth factor 4 [FGF4]), which can activate ES cell commitment and differentiation. The aim of ES cell culture is to expand the cell population while maintaining pluripotency. The researchers therefore cultured rat ES cells with leukemia inhibitory factor (LIF)-expressing mouse fibroblast feeder cells, in a medium containing two or three small-molecule inhibitors of pathways involved in ES cell commitment and differentiation, plus human LIF. (LIF supports proliferation of ES cells in an undifferentiated state.) This medium is known as 2i (for 2-inhibitors) or 3i medium.
Rat ES cells cultured in this manner expressed key molecular markers found in mouse ES cells. They also, when injected into blastocysts, can give rise to chimeric rats; i.e., they transmute their genomes into offspring. Such cultured rat ES cells thus are capable of being used to construct knockout rats.
In the 9 September 2010 issue of Nature, Dr. Qi-Long Ying (University of Southern California, Los Angeles CA) and his colleagues published the first study describing construction of a knockout rat strain via homologous recombination. (Dr. Ying, then at the University of Edinburgh, had been on the team led by Austin Smith that developed culture methods for rat ES cells.) This rat strain is a p53 gene knockout. The researchers designed a targeting vector to disrupt the p53 tumor suppressor gene via homologous recombination; the vector allowed for antibiotic selection for cells that had been successfully targeted. They transfected this vector into rat ES cells cultured in 2i medium, performed the antibiotic selection, and cultured the resistant cells. These cells were shown to have one of their two (since they were diploid) p53 genes disrupted. The researchers were able to routinely generate p53-targeted rat ES cells by this method. They also injected p53-targeted rat ES cells into rat blastocysts, transferred the blastocysts into pseudo-pregnant female rats, and obtained chimeric offspring. However, in the first studies, the p53-targeted rat ES cells exhibited low germline transmission efficiency.
In the mouse system, the failure of cultured ES cells to contribute to the germline is often caused by chromosomal abnormalities in the ES cells. This was also the case with the rat ES cells. In the case of mouse ES cell culture, cells with chromosomal abnormalities have a selective growth advantage over those with normal karyotypes. The smaller, slower-growing mouse ES cell clones tend to have normal karyotypes, and to give improved germline transmission. The researchers therefore subcloned their p53 gene-targeted rat ES cells, and selected for small, slower-growing subclones. These rat ES cell subclones were euploid. When injected into blastocysts, these rat ES cell clones gave rise to chimeric rats that the researchers further bred to generate homozygous p53 gene-targeted (i.e., p53 knockout, or p53 homozygous null) rats.
Using these methods, it should be possible to generate knockout rats for other genes routinely, including sophisticated knockouts such as tissue-specific gene knockouts.
Meanwhile, SAGE has generated p53 knockout rats, using its ZFN technology. As with the original p53 knockout mice, these rats develop normally, but are prone to development of spontaneous tumors. p53 knockout rats generated via homologous recombination should also be susceptible to spontaneous generation of tumors. However, as yet no data has been published. It remains to be seen which of these systems–p53 knockout mice or p53-knockout rats generated via either homologous recombination or ZFN editing, will be most useful in basic cancer research, or in such applications as carcinogenicity screening of compounds.
Why is the ability of researchers to generate knockout rats, as opposed to knockout mice, so important? The anatomy and physiology of the rat is closer to humans than is the mouse. There are also many rat models of complex human diseases (especially cardiovascular and metabolic diseases) that are better disease models than those based on inbred mouse strains. In addition, the larger size of the rat facilitates experimental procedures that involve surgery, getting blood samples for analysis, or isolation of specific cell populations. Researchers usually prefer rats over mice for physiological and nutritional studies, studies of psychiatric diseases, and in cases when a particular rat disease model is more applicable to a project than mouse strains. The rat is also widely used in preclinical efficacy and safety studies.
With respect to models for central nervous system (CNS) diseases, gene-targeted and transgenic rat models may be expected to be better than mouse models. The rat is more intelligent than the mouse, and has a bigger brain. Unlike mice, rats are sociable and easily trained. Moreover, there are some new rat models of cognition, which enable researchers to perform studies that they previously thought could only be done in nonhuman primates. And optogenetics technology, which allows researchers to engineer specific neurons so that their activity can be switched on or off with laser light, in order to dissect the role of these neurons in behavior, is being implemented in rats. These new developments, together with knockout and transgenic technologies, should allow researchers to develop new rat models of psychiatric diseases, as well as of neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. The lack of good animal models is a major factor in the high clinical attrition rate of CNS drugs, so new models are needed. There are of course no guarantees that novel rat models will help lower CNS drug attrition rates, but it is well worth trying these new approaches.
As we also discussed in Chapter 7 of Animal Models for Therapeutic Strategies, researchers are also interested in developing animal models based on mammalian species other than the mouse and the rat. We discussed methods for gene targeting by recombinant adeno-associated virus (rAAV) in pigs and ferrets in that chapter. In principle, ZFN editing technology could be also used to generate gene knockouts in mammalian species other than rodents. Moreover, the type of research done in the rat by Austin Smith, Qi-Long Ying, and their colleagues might be applied to developing culture conditions for ES cells of other mammalian species, which could set the stage for developing gene knockouts in these species via homologous recombination.