Knockout Rat - Early Methods

Early Methods

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.