Although the rat is an important model in studies of behaviour and physiology, it lags behind the mouse in the genetic popularity stakes, as rat embryonic stem cells cannot be used to produce gene-disrupted knockout lines.

However, this is about to change, as Zan et al., in Nature Biotechnology, describe an alternative approach, which they use to produce rat knockouts for the breast-cancer supressor genes Brca1 and Brca2 . Their innovative technique combines rat germline mutagenesis using N-ethyl-N-nitrosourea (ENU) with yeast truncation assays that screen for functional mutations in selected genes.

Initially, male rats are mutagenized and bred with wild-type females. The F1 pups are then screened for functional mutations in the gene of interest, using two related yeast truncation assays — one based on genomic DNA and the other on RNA that has been reverse transcribed to cDNA.

Whole-gene sequences or large fragments from the target gene are cloned into gap-repair vectors that are then transformed into competent Saccharomyces cerevisiae yIG397 cells. Once incorporated, the gene fragment is cloned behind the yeast Adh1 promoter and in front of the Ade2 reporter gene. Functional mutations that prevent the production of active Ade2 are identified by the growth of small red yeast colonies on selective medium; by contrast, yeast cells with functional Ade2 produce large white colonies.

Finally, heterozygous knockouts in the F1 progeny are bred to produce homozygotes. Direct sequencing of the N2 offspring of the knockout-founder males and wild-type females has verified the accuracy of the assays.

This ENU-induced mutation-screening method could produce unique gene-selected knockout rats that will extend our knowledge of the genetics that underlie human diseases and aid in drug development. So, in the future, rat models might give knockout mice a good run for their money.