INTRODUCTION OF THE NEGATIVE SELECTION MARKER INTO REPLACEMENT VECTORS BY A SINGLE LIGATION STEP

Gene targeting is a powerful method for introducing mutations into the genome of embryonic stem cells. The most widely used approach is the positive-negative selection method in which a gene encoding a negative selection marker is cloned into the replacement vector to obtain an e nrichment of properly targeted clones. Here, we present an alternative means to introduce any given negative selection marker at the ends of a replacement vector using a single ligation step, thereby avoiding l aborious cloning procedures. Our results demonstrate that this fast an d simple method consistently provides a high level of enrichment of ap propriately targeted clones.