An ENU-induced mutant archive for gene targeting in rats

To the editor: Although the laboratory rat is increasingly being used as a mammalian model in biomedical research, no technology exists thus far for the production of in vivo genetically engineered mutations equivalent to knockout or knock-in mice because of the lack of development of functional embryonic stem cells in this species. Rat spermatogonial stem cells may possibly have greater potential use for genetic engineering to produce gene-targeted rats1,2. The use of somatic cell nuclear transfer to develop cloned rats as an alternative to using embryonic stem cells has also been reported3, but it seems to be a very difficult technique to perform. At present, N-ethyl-Nnitrosourea (ENU) mutagenesis followed by a screening method to detect single-nucleotide substitutions within the targeted gene seems to be the most promising technology in rats, as previously reported by several groups4,5,6. The screening protocol, however, either a yeastbased screening assay4,5 or a highthroughput resequencing-based screening technique7, is expensive. In addition, gene-driven ENU mutagenesis approaches in rats are not a very efficient use of resources because most of the rats generated are usually discarded within a few weeks or months after the targeted genes have been screened owing to a lack of reliable sperm conservation and rederivation technologies. We have developed a new, efficient approach that combines two methods: a high-throughput, lowa b