Efficient repetitive alteration of the mouse Huntington's disease gene by management of background in the tag and exchange gene targeting strategy

The introduction of subtle mutations to predetermined locations in the mous e genome has aided in the assessment of gene function and the precise model ing of inherited disorders. Subtle mutations can be engineered into the mou se genome by the tag and exchange gene targeting strategy (Askew et al., 19 93; Stacey et al., 1994; Wu et al., 1994). This two-step method involves bo th a positive and a negative selection. The negative selection step typical ly generates a large amount of undesired background that may prevent the pr actical recovery of gene targeted clones (Vazquez et al., 1998). In this wo rk we describe a strategy to effectively manage this background by calculat ion of a tolerable level of background for a specific targeting event, pre- screening for clones with low background, subcloning and growth of cell lin es under selection. This strategy was used to repeatedly and efficiently al ter the mouse Huntington's disease homologue (Hdh) resulting in an average of 15 percent of the clones having the desired modification. Analysis of th e remaining background clones showed they arose de novo by a mechanism that involved physical loss of the marker rather than mutation or inactivation. We calculated the rate of loss of this marker as 8.3 x 10(-6) events/cell/ generation. We further show that the exchanged clones retained the capacity to contribute to the mouse germline demonstrating the utility of this stra tegy in the production of mouse lines with Hdh variants.