Genetic reporter system for oncogenic Igh-Myc translocations in mice

Abstract

The Myc-deregulating chromosomal T(12;15)(Igh-Myc) translocation, the hallmark mutation of inflammation- and interleukin 6-dependent mouse plasmacytoma (PCT), is the premier model of cancer-associated chromosomal translocations because it is the only translocation in mice that occurs spontaneously (B lymphocyte lineage) and with predictably high incidence (approximately 85% of PCT), and has a direct counterpart in humans: Burkitt lymphoma t(8;14)(q24;q32) translocation. Here, we report on the development of a genetic system for the detection of T(12;15)(Igh-Myc) translocations in plasma cells of a mouse strain in which an enhanced green fluorescent protein (GFP)-encoding reporter gene has been targeted to Myc. Four of the PCTs that developed in the newly generated translocation reporter mice, designated iGFP(5'Myc), expressed GFP consequent to naturally occurring T(12;15) translocation. GFP expression did not interfere with tumor development or the deregulation of Myc on derivative 12 of translocation, der (12), because the reporter gene was allocated to the reciprocal product of translocation, der (15). Although the described reporter gene approach requires refinement before T(12;15) translocations can be quantitatively detected in vivo, including in B lymphocyte lineage cells that have not yet completed malignant transformation, our findings provide proof of principle that reporter gene tagging of oncogenes in gene-targeted mice can be used to elucidate unresolved questions on the occurrence, distribution and trafficking of cells that have acquired cancer-causing chromosomal translocations of great relevance for humans.

Figure 1

Genetic system for detection of the T(12;15) translocation in B-lineage cells of iGFP5′Myc gene-insertion mice. Shown above are schematic representations of the normal mouse Myc locus on chromosome 15, and of the targeted Myc locus carrying the inserted V_H-GFP-Neo gene in iGFP^5′Myc knock-in mice. The non-coding first exon of Myc is depicted by a red box, with the P1 and P2 Myc promoters indicated by two red arrows pointing right. The coding region of Myc (exons 2 and 3) and the 3′ untranslated region of Myc exon 3 are depicted by two pink boxes and one white box, respectively. The 1.6-kb first intron of Myc is not drawn to scale (as indicated by the short, oblique double line). The transcriptional orientations of the inserted GFP and Neo genes are indicated by colored arrows at transcriptional start sites. Shown in center is a schematic representation of the Igh locus on chromosome 12. The Igh variable (V_H), diversity (D) and joining (J_H) regions are represented by thick vertical lines labeled as such. The Igh constant region (C_H), which is flanked by the intronic heavy-chain enhancer (Eµ) and the 3′ Cα heavy-chain enhancer (Eα; indicated by two black diamonds), is only partially represented by four C_H genes: Cµ, Cα, Cγ2b and Cα (white, labeled boxes). The corresponding switch regions are indicated by black dots (except in the case of Cδ, which does not have a canonical switch region). Four additional genes in the mouse C_H cluster (Cγ1, Cγ2a, Cγ3, Cε) are not shown. The Igh locus and the targeted Myc locus are aligned at a crossover site typically used to generate the T(12;15) translocation: the switch µ region on chromosome 12 and the first intron of Myc on chromosome 15. This is denoted by a cross-labeled alignment with Sµ. The actual site of DNA double-strand breakage and reciprocal transchromosomal recombination is indicated by a vertical, dashed line and an arrow-labeled T(12;15) translocation. Shown at bottom are schematic representations of the reciprocal products of the translocation: der(15) and der(12). Juxtaposition of Eµ to the V_H promoter of the GFP gene leads to the expression of GFP on der(15), as indicated by a thick green arrow pointing left. Annealing sites for PCR primers in Igh, Myc and V_H-GFP used to detect reciprocal Igh–Myc junctions on der(12) and Igh–Myc–GFP junctions on der(15) are indicated by horizontal arrows that are colored black, red and green, respectively.

Figure 2

The iGFP^5′Myc translocation reporter does not affect the genetic susceptibility of mice to plasmacytoma development. Depicted are onset and incidence of inflammation (pristane)-induced peritoneal plasmacytoma (PCT) in double-transgenic C.iGFP^5′Myc/BCL2 mice (squares) and single-transgenic C.BCL2 mice (circles). In both strains, the BCL2 transgene accelerated PCT development with the same efficiency as observed in a previous study (Silva et al., 2003). Single transgenic C.iGFP^5′Myc mice (triangles pointing up) and non-transgenic littermates (triangles pointing down), in which tumor development began just before termination of the study, on day 160 after tumor induction with pristane were included as controls. In all mice, PCTs were detected by finding neoplastic plasma cells in ascites-cell specimens that had been stained according to Wright–Giemsa.

Figure 3

Plasma-cell tumors in iGFP^5’Myc mice express GFP consequent to T(12;15) translocation. (a) GFP expression in tumor cells, as detected by flow cytometry. Shown are scatter plots of ascites cells from mice bearing PCT8 (left, negative control) and PCT2 (right). Cells were treated with an Fc blocker (antibody to CD16/CD32) and stained with an antibody to B220 (CD45R; RA3–6B2) or CD138 (281–2). Isotype-matched antibodies of unrelated specificity confirmed the specificity of the B220 and CD138 signals. Cells were analyzed on a Beckman Coulter FC 500 instrument (Beckman Coulter Inc., Fullerton, CA, USA) using FlowJo software. Note the substantially expanded CD138⁺GFP⁻ population of tumor cells (indicated by grey rectangle; 9% of all cells) that co-existed with the predominant CD138⁺GFP⁺ tumor cell clone (24%) in the PCT2-harboring mouse. (b) GFP expression in T(12;15)⁺CD138⁺ tumor cells, as detected by immunofluorescence microscopy. Shown in the top and center rows are confocal microscopy images of serial tissue sections of a peritoneal granuloma that was labeled with PE-conjugated antibody to CD138 or B220, followed by counterstaining with DAPI (blue). These images are shown at low magnification. Adjacent to the granuloma (upper half of each image) is a piece of the mesenteric lymph node (MLN, lower half of each image). The three images in the top row show that plasma cells expressing GFP (left panel) and CD138 (center panel) had infiltrated the granuloma in large numbers, but were rare (scattered) in the MLN. The images in the center row reveal B220⁺GFP⁻ B cells that populate the MLN but not the granuloma. The bottom-row images present the CD138⁺GFP⁻ tumor cells included in the white square in the upper left-hand panel taken at higher magnification. (c) Presence of the T(12;15), as detected by FISH. Metaphase chromosome spread of PCT 1 hybridized to FISH probes for Myc (FITC, green, Chr 15) and Igh (Cy5, red, Chr 12). der(12) is indicated by co-localization of FISH signals, whereas der(15) is visualized by the weak Myc signal to the upper right of the image. Chromosomes were counter-stained with DAPI (blue). (d) Rearrangement of GFP-targeted Myc allele with Igh as detected by PCR analysis. Shown are der(15)-typical GFP–Myc–Igh junction fragments (red rectangles) detected in PCT1–4 and PCT10–11 by PCR analysis of genomic DNA. Junction fragments were size fractionated in an agarose gel and confirmed to be clonotypic by DNA sequence analysis of the underlying DNA recombination (not shown). PCR fragments outside of the red boxes, the most prominent of which are indicated by grey arrows pointing right, are either artifacts or bona fide translocation junction fragments indicating the clonal diversity of incipient PCTs in strain C mice. Size markers (kb) are indicated.

Figure 4

Flow cytometric detection of GFP⁺ splenic B cells from C.iGFP^5′Myc mice. Viable mononuclear spleen cell suspensions from unimmunized BALB/c (C) control, unimmunized C.iGFP^5′Myc, or SRBC hyperimmunized C.iGFP^5′Myc mice were prepared and stained with anti-B220 and anti-CD19 mAbs, as described in Supplementary Methods. Cells were analyzed on a BD FACSCanto with 3 × 10⁶ events collected per sample to ensure detection of rare events. Using FlowJo analysis software, viable single cells were carefully selected using forward versus side scatter, followed by forward scatter versus forward scatter pulse width gating. (a) Representative plots from unimmunized C, unimmunized C.iGFP^5′Myc and hyperimmunized C.iGFP^5′Myc mice are shown. Representative CD19 versus GFP bivariate plots (right panels) derived from the B220⁺ gate (left panels) are illustrated. The square gates used to calculate the frequency of B220⁺CD19⁺GFP⁺ events are shown on the CD19 versus GFP plots. Note that CD19⁻ plasmacytoid dendritic cells (pDC) are present within the B220⁺ gate. Their identity was confirmed with CD11c staining (data not shown). (b) Graphs represent the frequency of CD19⁺GFP⁺ B cells within the B220⁺ gated population (left panel) and the total number of B220⁺CD19⁺GFP⁺ cells per spleen (right panel). BALB, unimmunized C control; iGFP^5′Myc, unimmunized C.iGFP^5′Myc; iGFP^5′Myc immune, hyperimmunized C.iGFP^5′Myc. Values obtained from C control mice represent background levels. Bar graphs = mean ± s.d.; n = 3–4 per group.