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Recent studies have suggested that targeted mutations that retain the PGK—Neo cassette may yield unexpected phenotypes due to the altered expression of neighboring genes 1 ; the mechanism is unknown. Remarkably, the insertion of PGK—Neo abrogrates the expression of multiple globin genes downstream from the cassette. Deletion of the selectable marker cassette results in restoration of LCR activity, suggesting that the cassette disrupts normal interactions between the LCR and downstream regulatory elements.
These examples underscore the unpredictable phenotypes that can be caused by retained PGK—Neo cassettes. Recent studies have identified a cluster of closely linked hematopoietic serine protease genes granzymes that are tightly regulated and expressed specifically in activated cytotoxic lymphocytes 19 — Our laboratory has shown that cytotoxic T lymphocytes CTLs , natural killer NK cells, and lymphokine-activated killer LAK cells require one of these enzymes, granzyme B, for the rapid induction of apoptosis in allogeneic target cells The granzyme B loci of humans and mice are found in syntenic regions of chromosome 14 25 — The human locus is composed of four functional genes: granzymes B and H, cathepsin G, and mast cell chymase.
Cathepsin G is expressed in promyelocytes 29 , and chymase is expressed preferentially in mast cells Previous studies have suggested that mouse granzymes B—G and cathepsin G are also tightly clustered 26 , 28 , In this report, we show that insertion of PGK—Neo into the murine granzyme B gene abrogates the expression of several granzyme genes within the granzyme B gene cluster.
In addition, we have learned that PGK—Neo can be governed by regulatory elements in the domains into which it is inserted. These observations have important implications for the interpretation of phenotypes in knockout animals where the selectable marker is retained within the targeted locus. To screen for the presence of each gene on the overlapping clones, the following strategies were used: granzyme B was screened by Southern blot analysis using a 0.
Granzymes D, E, F, and G and MMCP-2 were screened by PCR using exon-specific primers derived from sequences in exon 3—4 and exon 4—5 splice junctions all primer sequences are available upon request from the authors. Total splenocytes were activated in a one-way mixed lymphocyte reaction MLR as described Generation of LAK cells was also performed as described Total cellular RNA was prepared and analyzed by S1 nuclease protection as described Granzyme D- and E-specific probes were generated using primers that amplified a genomic fragment containing the intron 4—exon 5 splice junction.
The protected probe fragment sizes are indicated under the probes in Fig. The probe for PGK—Neo was prepared from a 0. Mouse granzyme B cluster. The human granzyme B cluster, located on chromosome 14q The relative position of granzymes E and G on overlapping clones was determined by PCR using exon-specific primers. 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. In mice, seven granzymes A—G have been described to date. The granzyme A gene resides on chromosome 13 31 , and the granzyme B cluster is found on chromosome 14 Each PCR product was subcloned and sequenced to confirm its identity.
Granzymes D, E, and G to our knowledge, whose gene structure has not been reported were sequenced completely to determine their genomic organizations Fig. The blots were hybridized sequentially with probes specific for granzymes B, C, F, and D data not shown. Granzyme C lies approximately 23 kb downstream from granzyme B and 25 kb upstream from granzyme F.
Granzymes F and D colocalized on the same kb Nru I fragment data not shown. MMCP-1, -4, and -5, which are known to reside in the chymase gene cluster on chromosome 14 34 , presumably lie further downstream.
The transcriptional orientation of each gene in the cluster has not yet been determined; all genes in the human cluster are in the same orientation Fig. Using a series of specific S1 probes designed to discriminate among the highly related granzyme transcripts, we examined MLR RNA samples for the expression of granzymes in the cluster.
Expression of granzymes A—G in activated lymphocytes. We next examined granzyme gene expression in LAK cells derived from mice containing targeted mutations in either granzyme B or cathepsin G. PGK—Neo is transcribed in the same direction as the granzyme B gene. RNA and protein analyses have confirmed that this mutation is null for cathepsin G D. The proliferative potential of splenocytes in response to high-dose IL-2 was not affected by either mutation data not shown. We assayed granzyme A mRNA levels since this gene is located on chromosome 13 as an indicator of adequate lymphocyte activation.
The granzyme B mutation did not alter cathepsin G expression in the bone marrow ref. We determined that the reduction in granzyme C, F, D, and G expression was not due to gross alterations in these genes, since Southern blot analyses revealed that all genes were present and nonrearranged data not shown.
Equivalent levels of granzyme A mRNA in all three samples indicates that all samples were similarly activated. These experiments were performed three times, yielding identical results.
Granzyme G and E panels were exposed for 72 h. The granzyme B mutation does not affect expression of cathepsin G in marrow lane 6 , as reported These experiments were repeated four times with identical results.
We next wanted to determine whether PGK—Neo expression was affected by its position in the granzyme B cluster. In these studies, we have established that the PGK—Neo cassette can severely affect the expression of several genes within a multigene cluster. In this report, we have learned that a PGK—Neo cassette located in the granzyme B gene disrupts the expression of multiple granzyme genes downstream from granzyme B in LAK cells. However, a PGK—Neo cassette located in the cathepsin G gene just downstream from the granzyme genes caused minimal alterations in the expression of the upstream granzymes, suggesting that it lies within a separate regulatory domain within this multigene cluster.
Thus, these results have implications for how PGK—Neo cassettes can potentially alter the expression of multiple genes within a locus and suggests a mechanism for how that disregulation might occur. However, the granzyme B mutation had no effect on the expression of cathepsin G, a promyelocyte-specific gene that lies 30—50 kb downstream from the minimally expressed granzyme E gene.
These results imply that the active granzyme domain may be regulated by a common regulatory element and that the insertion of PGK—Neo disrupts a normal interaction between this putative element and the downstream genes Fig.
Since cathepsin G is expressed predominantly in myeloid precursors, this gene may lie in a separate domain within the locus and may have separate elements that regulate its expression. Model of competition between the PGK—Neo cassette and the individual granzyme promoters for a productive interaction with a putative LCR. This disruption is position-specific, since insertion of PGK—Neo into the cathepsin G gene has minimal effects on granzyme gene expression.
These results suggest that the PGK—Neo cassette is captured by the regulatory elements of the granzyme B gene or domain, causing PGK—Neo to adopt the regulatory behavior of the granzyme B gene itself. CTLs derived from these mice have a profound defect in their ability to rapidly induce apoptosis in allogeneic target cells.
Since the CTLs derived from MLRs predominantly express granzymes A and B, we strongly suspect that the defect in the induction of apoptosis is due to the lack of granzyme B itself. However, since LAK cells clearly express granzymes B, C, D, and F and since all of these genes are disregulated by the mutation, we do not know whether the cytotoxic defect observed is due to the loss of granzyme B or the loss of other granzymes in the cluster. To discriminate between these possibilities, we are creating mice with the same granzyme B mutation, but with the PGK—Neo cassette removed; we are also inserting the PGK—Neo cassette between granzymes B and C to determine whether all of the downstream genes can be disrupted by this mutation.
These results demonstrate the potential of a PGK—Neo cassette to mitigate the expression of several genes within a multigene locus, making interpretation of specific phenotypes potentially difficult to ascertain. We thank Jeff Milbrandt and Mark Groudine for helpful suggestions and advice.
Robin Wesselschmidt and Pam Goda provided expert animal care and technical assistance. We thank Dr. Nancy Reidelberger provided expert assistance with preparation of the manuscript. Data deposition: The sequences reported in this paper have been deposited in the GenBank data base accession nos.
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Long-range disruption of gene expression by a selectable marker cassette.
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