Long-range disruption of gene expression by a selectable marker cassette

Abstract

Recent studies have suggested that the retention of selectable marker cassettes (like PGK-Neo, in which a hybrid gene consisting of the phosphoglycerate 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 locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the ␤-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 genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK-Neo cassette into the ␤-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation 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 long distance effects are unknown, the expression of PGK-Neo can be ''captured'' by the regulatory domain into which it is inserted. These results suggest that the PGK-Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.

Mutations of the PU.1 Ets domain are specifically associated with murine radiation-induced, but not human therapy-related, acute myeloid leukaemia

“…Similar to analysis of an earlier knockout PU.1 model , AML does not occur in PU.1 knockdown mice showing complete or 50% loss of PU.1 expression (Rosenbauer et al, 2004). Although the study by Rosenbauer et al (2004) has shown a direct link between AML and PU.1, the mechanism proposed remains open to some doubt as the Neo used to replace the -14 kb upstream regulatory region of PU.1 may effect the expression of genes several 100 kb away (Pham et al, 1996). In the present study, mutations in the conserved, and potential regulatory regions, upstream of PU.1 were not found in human AML.…”

Analysis of congenital hypomyelinating Egr2 Lo/Lo nerves identifies Sox2 as an inhibitor of Schwann cell differentiation and myelination

“…In attempting to introduce specific point mutants into the Egr2 locus, we found that insertion of a PGK-Neo cassette into the first intron of Egr2 generated a hypomorphic Egr2 allele (Egr2 Lo ). The decreased expression from this mutant allele is presumably due to the introduction of the PGK promoter, as has been observed at other loci (13). Mice heterozygous (Egr2 ϩ/Lo ) for the hypomorphic allele were overtly normal, and homozygous mice (Egr2 Lo/Lo ) were produced at expected Mendelian ratios.…”

Abnormalities of developmental cell death in Dad1‐deficient mice

“…In the latter case, it is possible that a small number of functional transcripts still remain, making heterozygous phenotypes that are somewhat milder than those we obtained. If we consider that the granzyme B cluster, which consists of granzyme B-G, cathepsin G and mouse mast cell protease 2, exists at nearly the same chromosomal location as the T-cell receptor a/d gene (and the Dad1 gene), then it is likely that genes which have some relation to apoptosis may comprise a functional complex around the Dad1 locus (Pham et al 1996). Further investigations into the Dad1 genomic structure and function would help us to fully understand the phenotypes of the Dad1 mutant.…”