Long-range disruption of gene expression by a selectable marker cassette - PubMed (original) (raw)

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

C T Pham et al. Proc Natl Acad Sci U S A. 1996.

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 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 genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK-Neo cassette into the beta-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.

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Figures

Figure 1

Figure 1

Mouse granzyme B cluster. The human granzyme B cluster, located on chromosome 14q11.2, has been described elsewhere (27). (A) Overlapping murine P1 and BAC clones were characterized by rare-cutting restriction analysis, and the relative position of each granzyme gene was determined by Southern blot analysis using specific probes for granzymes B, C, D, and F, CG, and MMCP-2. The relative position of granzymes E and G on overlapping clones was determined by PCR using exon-specific primers. The sizes of the P1 and BAC clones were determined by CHEF gel analysis. Slashes between genes indicate approximate distances. (B) The genomic organization of the granzymes and cathepsin G. Coding sequences are designated as solid boxes and introns are designated as open boxes. Specific probes for S1 nuclease protection assays and the lengths (in nucleotides) of the fragments protected by correctly spliced mRNAs are shown for each gene. Asterisks designate a newly defined gene structure.

Figure 2

Figure 2

Expression of granzymes A–G in activated lymphocytes. (A) Twenty micrograms of total cellular RNA derived from a day 5 MLR culture was hybridized with a panel of specific probes for granzymes A–G (see Fig. 1). The designated granzyme probe and β2-microglobulin probe were cohybridized with each RNA sample, and S1 analysis was performed. Note that mRNAs encoding granzymes A and B are most abundant, with small amounts of granzyme C detected on day 5. Similar results were obtained using RNA obtained from day 2–5 MLR (data not shown). (B) LAK cells were generated by culturing splenocytes in the presence of high-dose IL-2 for 10 days. Twenty micrograms of total RNA was hybridized with specific probes for granzymes A–G (see Fig. 1). (C) Total RNA derived from the NK tumor cell line NK 3.1 (CD3−, NK1.1+, maintained in recombinant human IL-2 at 500 units/ml), was characterized using specific probes for granzymes A–G. Note that LAK cells and NK 3.1 cells both contain abundant levels of granzyme A, B, C, D, and F mRNAs.

Figure 3

Figure 3

Expression of granzymes in LAK cells derived from wild-type (WT), granzyme B −/−, and cathepsin G −/− mice. LAK cell total RNA (20 μg), generated from WT, B −/−, and cathepsin G −/− mice was analyzed for the expression of granzymes A–G using S1 nuclease protection assays. Equivalent levels of granzyme A mRNA in all three samples indicates that all samples were similarly activated. Note that levels of granzymes C, F, and D mRNAs are substantially reduced in LAK cells derived from granzyme B −/− mice. These experiments were performed three times, yielding identical results. Autoradiograms for granzyme A, B, C, F, and D panels were exposed overnight. Granzyme G and E panels were exposed for 72 h.

Figure 4

Figure 4

Expression of PGK–Neo in wild-type (WT), granzyme B −/−, cathepsin G −/−, and HS-3 −/− mice. RNA (15 μg) derived from resting spleen, MLR, and bone marrow from each animal was analyzed using S1 nuclease protection assays; each mRNA sample was cohybridized with probes for granzyme B, cathepsin G, PGK–Neo, and β2-microglobulin. Note that abundant, correctly initiated PGK–Neo mRNA is detected in MLR lymphocytes (or LAK cells, data not shown) only when the cassette is located within the granzyme B gene (lane 5 versus lanes 8 and 11). PGK–Neo is detected in the spleen and bone marrow of mice containing the cassette in the murine β-globin LCR (lanes 10 and 12); both of these organs contain erythroid precursors in adult mice. PGK–Neo mRNA is not detected in the marrow of the mice containing the cathepsin G −/− mutation. The granzyme B mutation does not affect expression of cathepsin G in marrow (lane 6), as reported (24). These experiments were repeated four times with identical results.

Figure 5

Figure 5

Model of competition between the PGK–Neo cassette and the individual granzyme promoters for a productive interaction with a putative LCR. Insertion of PGK–Neo may disrupt normal interactions between the putative LCR and the granzyme genes, leading to activation of the PGK–Neo gene and inactivation of multiple genes within the locus. This disruption is position-specific, since insertion of PGK–Neo into the cathepsin G gene has minimal effects on granzyme gene expression.

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