Knockout rat

A knockout rat is a genetically engineered rat with a single gene turned off through a targeted mutation (gene trapping) used for academic and pharmaceutical research. Knockout rats can mimic human diseases and are important tools for studying gene function (functional genomics) and for drug discovery and development. The production of knockout rats was not economically or technically feasible until 2008. A knockout rat is a genetically engineered rat with a single gene turned off through a targeted mutation (gene trapping) used for academic and pharmaceutical research. Knockout rats can mimic human diseases and are important tools for studying gene function (functional genomics) and for drug discovery and development. The production of knockout rats was not economically or technically feasible until 2008. Technology developed through funding from the National Institutes of Health (NIH) and work accomplished by the members of the Knock Out Rat Consortium (KORC) led to cost-effective methods to create knockout rats. The importance of developing the rat as a more versatile tool for human health research is evidenced by the $120 million investment made by the NIH via the Rat Genome Sequencing Project Consortium, resulting in the draft sequence of a laboratory strain of the brown or Norway rat (Rattus norvegicus). Additional developments with zinc finger nuclease technology in 2009 led to the first knockout rat with targeted, germline-transmitted mutations. Knockout rat disease models for Parkinson's, Alzheimer's, hypertension, and diabetes using zinc-finger nuclease technology are being commercialized by SAGE Labs. Mice, rats, and humans share all but approximately 1% of each other's genes making rodents good model organisms for studying human gene function. Both mice and rats are relatively small, easily handled, have a short generation time, and are genetically inbred. While mice have proven to be a useful rodent model and techniques have been developed for routine disruption of their genes, in many circumstances rats are considered a superior laboratory animal for studying and modeling human disease. Rats are physiologically more similar to humans than are mice. For example, rats have a heart rate more similar to that of humans, while mice have a heart rate five to ten times as fast. It is widely believed that the rat is a better model than the mouse for human cardiovascular disease, diabetes, arthritis, and many autoimmune, neurological, behavioral, and addiction disorders. In addition, rat models are superior to mouse models for testing the pharmacodynamics and toxicity of potential therapeutic compounds, partially because the number and type of many of their detoxifying enzymes are very similar to those in humans. Their larger size makes rats more conducive to study by instrumentation, and also facilitates manipulation such as blood sampling, nerve conduction, and performing surgeries. Techniques for genetic manipulation are available in the mouse, which is commonly used to model human disease. Although published knockouts exist for approximately 60% of mouse genes, a large majority of common human diseases do not have a knockout mouse model. Knockout rat models are an alternative to mice that may enable the creation of new gene disruptions that are unavailable in the mouse. Knockout rat models can also complement existing transgenic mouse models. Comparing mouse and rat mutants can facilitate the distinction between rodent-specific and general mammalian phenotypes. Rat models have been used to advance many areas of medical research, including cardiovascular disease, psychiatric disorders (studies of behavioral intervention and addiction), neural regeneration, diabetes, transplantation, autoimmune disorders (rheumatoid arthritis), cancer, and wound & bone healing. While the completion of the rat genome sequence provides very key information, how these diseases relate to gene function requires an efficient method to create knockout rat models in which specific genomic sequences are manipulated.Most techniques for genetic manipulation, including random mutagenesis with a gene trap (retroviral-based and non-retroviral-based), gene knock-outs/knock-ins, and conditional mutations, depend upon the culture and manipulation of embryonic stem (ES) cells. Rat ES cells were only recently isolated and no demonstration of gene modification in them has been reported. Consequently, many genetic manipulation techniques widely used in the mouse are not possible in the rat. Until the commercial development of mobile DNA technology in 2007 and zinc-finger nuclease technology in 2009, there were only two technologies that could be used to produce rat models of human disease: cloning and chemical mutagenesis using N-ethyl-N-nitrosourea (ENU). Although cloning by somatic cell nuclear transfer (SCNT) could theoretically be used to create rats with specific mutations by mutating somatic cells, and then using these cells for SCNT, this approach has not been used successfully to create knockout rats. One problem with this strategy is that SCNT is extremely inefficient. The first published attempt had a success rate of less than 1%. Alternatively, ENU mutagenesis is a common random mutagenesis gene knockout strategy in the mouse that can also be used in the rat. ENU mutagenesis involves using a chemical, N-ethyl-N-nitrosourea (ENU), to create single base changes in the genome. ENU transfers its ethyl group to oxygen or nitrogen radicals in DNA, resulting in mis-pairing and base pair substitution. Mutant animals can be produced by injecting a male mouse with ENU, and breeding with a wild type female to produce mutant offspring. ENU mutagenesis creates a high frequency of random mutations, with approximately one base pair change in any given gene in every 200-700 gametes. Despite its high mutagenicity, the physical penetration of ENU is limited and only about 500 genes are mutated for each male and a very small number of the total mutations have an observable phenotype. Thousands of mutations typically need to be created in a single animal in order to generate one novel phenotype. Despite recent improvements in ENU technology, mapping mutations responsible for a particular phenotype is typically difficult and time-consuming. Neutral mutations must be separated from causative mutations, via extensive breeding. ENU and cloning methods are simply inefficient for creating and mapping gene knockouts in rats for the creation of new models of human disease. Through 2007, the largest rat ENU mutagenesis project to date run by the Medical College of Wisconsin was able to produce only 9 knockout rat lines in a period of five years at an average cost of $200,000 per knockout line. Although some companies are still pursuing this strategy, the Medical College of Wisconsin has switched to a more efficient and commercially viable method using mobile DNA and CompoZr ZFN technology. Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) are engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations. Double strand breaks are important for site-specific mutagenesis in that they stimulate the cell’s natural DNA-repair processes, namely homologous recombination and non-homologous end joining. When the cell uses the non-homologous end joining pathway to repair the double-strand break, the inherent inaccuracy of the repair often generates precisely targeted mutations. This results in embryos with targeted gene knockout. Standard microinjection techniques allow this technology to make knockout rats in 4–6 months. A major advantage of ZFN- and TALEN-mediated gene knockout relative to the use of mobile DNA is that a particular gene can be uniquely and specifically targeted for knockout. In contrast, knockouts made using mobile DNA technology are random and are therefore unlikely to target the gene of interest.