LONG-RANGE DISRUPTION OF GENE-EXPRESSION BY A SELECTABLE MARKER CASSETTE

Recent studies have suggested that the retention of selectable marker cassettes (like PGK-Neo, in which a hybrid gene consisting of the phos phoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in ''knockout'' mice due to disruption of expression of neighboring genes within a lo cus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the beta-like globin gene cluster, The insertion of PGK-Neo into the granzyme B gene, the most 5' gene in the granzyme B gene cluster, severely reduced the normal expression of multiple ge nes within the locus, even at distances greater than 100 Iib from the mutation, Similarly, the insertion of a PGK-Neo cassette into the beta -globin locus control region (LCR) abrogates the expression of multipl e globin genes downstream from the cassette, In contrast, a targeted m utation of the promyelocyte-specific cathepsin G gene (which lies just 3' to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression, Although the mechanism of these lon g distance effects are unknown, the expression of PGK-Neo can be ''cap tured'' by the regulatory domain into which it is inserted. These resu lts suggest that the PGK-Neo cassette can interact productively with l ocus control regions and thereby disrupt normal interactions between l ocal and long-distance regulatory regions within a tissue-specific dom ain.