Inter-chromosomal recombination of Mll and Af9 genes mediated by cre-loxP in mouse development

Abstract

Chromosomal translocations are crucial events in the aetiology of many leukaemias, lymphomas and sarcomas, resulting in enforced oncogene expression or the creation of novel fusion genes. The study of the biological outcome of such events ideally requires recapitulation of the tissue specificity and timing of the chromosomal translocation itself. We have used the Cre-loxP system of phage P1 to induce de novo Mll-Af9 chromosomal recombination during mouse development. loxP sites were introduced into the Mll and Af9 genes on chromosomes 9 and 4, respectively, and mice carrying these alleles were crossed with mice expressing Cre recombinase. A resulting Mll-Af9 fusion gene was detected whose transcription and splicing were verified. Thus, programmed interchromosomal recombination can be achieved in mice. This approach should allow the design of mouse models of tumorigenesis with greater biological relevance than those available at present.

Fig. 1. Strategy for Cre-loxP-mediated recombination between the Mll and Af9 genes on chromosomes 9 and 4. (A) Targeted Mll and Af9 alleles indicating loxP sites (large arrowheads) within the introns corresponding to the breakpoint regions in humans. Cre-mediated recombination between loxP sites results in inter-chromosomal recombination and the generation of two fusion genes. Following transcription and RNA splicing, an in-frame Mll–Af9 mRNA is made. (B) Nested PCR primers for the detection of the Mll–Af9 fusion gene. (C) Nested PCR primers for RT–PCR detection of Mll–Af9 fusion mRNA. (D) Expected sequence at the junction of the Mll–Af9 fusion chromosome. (E) Expected sequence at the junction of the Mll–Af9 fusion mRNA.

Fig. 2. Targeting strategy for the insertion of loxP sites into Mll and Af9 genes via homologous recombination in mouse ES cells. (A) Genomic map of mouse Mll around exon 8 (line 1). Targeting vector pMll-loxP-hygro, with a loxP-hygro cassette inserted at a BglII site (line 2). Mutant Mll allele after homologous recombination (line 3). (B) Filter hybridization of targeted ES cell DNA. The 3′ Mll probe detected a 6 kb KpnI fragment wt allele and an 8 kb fragment mutant allele (left). The latter hybridized in targeted DNA with a probe for the inserted hygromycin gene (right); only a single hygromycin band was observed. (C) Genomic Af9 segment containing the intron analogous to the human AF9 translocation breakpoint region (line 1). Targeting vector pAf9-puro-loxP has a puromycin-loxP cassette inserted into an XbaI site (line 2). Mutant Af9 allele after homologous recombination (line 3). (D) Filter hybridization analysis of ES clone DNA. The 3′ Af9 probe detected an 11 kb SphI wt allele, and an 8 kb fragment mutant allele (left). The same band hybridized in targeted DNA with a probe for the inserted puromycin fragment (right); a single puromycin insertion was detected. wt, wild type; Mlox, Mll-targeted ES cell DNA; Alox, Af9-targeted ES cell DNA; tk, HSV thymidine kinase; B, BamHI; Bg, BglII; Bx, BstxI; H, HindIII; Kp, KpnI; N, NotI; R, EcoRI; RV, EcoRV; S, SacI; Sp, SphI; Xb, XbaI. Filled boxes represent exons. loxP sites are shown as arrowheads.

Fig. 3. Detection of Cre-loxP-mediated recombination between murine Mll and Af9 genes. (A) Detection of recombination in ES cells. Cells carrying both Mlox- and Alox-targeted alleles (MALX10) were subjected to transient expression of Cre recombinase (MALX-Cre). Nested PCR on genomic DNA (left) or RT–PCR on cDNA (right) was carried out with primers spanning the Mll–Af9 recombination junctions (Figure 1). Control reactions were carried out with either no DNA template (H₂O) or on genomic DNA from untransfected CCB cells or from the untransfected MALX10 cells. (B) Detection of recombination between Mll and Af9 genes in mouse tissues. Nested PCR analysis was carried out on genomic DNA from a variety of tissues from an Mlox–Alox control mouse (left) and from an Mlox–Alox–Cre animal with the deleter genotype (right). Reactions lacking template (H₂O) served as negative controls. Fragment sizes were estimated by co-electrophoresis of φX174 DNA cleaved with HaeIII.

Fig. 4. Detection of Mll–Af9 fusion RNA from tissues of Mlox–Alox–Cre mice. (A) Mll–Af9–specific nested RT–PCR analysis of cDNA generated from tissue RNA samples of Mlox–Alox or Mlox–Alox–Cre mice. (B) Detection of Cre expression by RT–PCR using cDNA template. (C) Actin-specific RT–PCR on the various cDNA populations.

Fig. 5. Detection of Mll–Af9 fusion in brain RNA of Mlox–Alox–Cre mice. (A) Mll–Af9–specific RT–PCR on cDNA generated from brain RNA of 10 Mlox–Alox–Cre (numbered 1–10) and two Mlox–Alox mice (numbered N1 and N2). (B) Actin-specific RT–PCR on the various cDNA populations.