DOUBLE REPLACEMENT GENE TARGETING FOR THE PRODUCTION OF A SERIES OF MOUSE STRAINS WITH DIFFERENT PRION PROTEIN GENE ALTERATIONS

We have developed a double replacement gene targeting strategy which e nables the production of a series of mouse strains bearing different s ubtle alterations to endogenous genes. This is a two-step process in w hich a region of the gene of interest is first replaced with a selecta ble marker to produce an inactivated allele, which is then re-targeted with a second vector to reconstruct the inactivated allele, concomita ntly introducing an engineered mutation. Five independent embryonic st em cell lines have been produced bearing different targeted alteration s to the prion protein gene, including one which raises the level of e xpression. We have constructed mice bearing the codon 101 proline to l eucine substitution linked to the human familial prion disease, Gerstm ann-Straussler-Scheinker syndrome. We anticipate that this procedure w ill have applications to the study of human inherited diseases and the development of therapies.