RAPID CONSTRUCTION IN YEAST OF COMPLEX TARGETING VECTORS FOR GENE MANIPULATION IN THE MOUSE

Targeting vectors for embryonic stem (ES) cells typically contain a mo use gene segment of >7 kb with the neo gene inserted for positive sele ction of the targeting event, More complex targeting vectors carry add itional genetic elements (e.g. lacZ, loxP, point mutations), Here we u se homologous recombination in yeast to construct targeting vectors fo r the Incorporation of genetic elements (GEs) into mouse genes, The pr ecise insertion of GEs into any position of a mouse gene segment clone d in an Escherichia coli/yeast shuttle vector is directed by short rec ombinogenic arms (RAs) flanking the GEs, In this way, complex targetin g vectors can be engineered with considerable ease and speed, obviatin g extensive gene mapping in search for suitable restriction sites.