Inducible chromosomal translocation of AML1 and ETO genes through Cre/loxP-mediated recombination in the mouse

Abstract

Transgenic mice have been used to explore the role of chromosomal translocations in the genesis of tumors. But none of these efforts has actually involved induction of a translocation in vivo. Here we report the use of Cre recombinase to replicate in vivo the t(8;21) translocation found in human acute myeloid leukemia (AML). As in the human tumors, the murine translocation fuses the genes AML1 and ETO. We used homologous recombination to place loxP sites at loci that were syntenic with the break points for the human translocation. Cre activity was provided in mice by a transgene under the control of the Nestin promoter, or in cultured B cells by infecting with a retroviral vector encoding Cre. In both instances, Cre activity mediated interchromosomal translocations that fused the AML1 and ETO genes. Thus, reciprocal chromosomal translocations that closely resemble rearrangements found in human cancers can be achieved in mice.

Fig. 1. Scheme for induction of chromosomal translocation by Cre. Mice containing a loxP site in intron 1 of the ETO gene on chromosome 4 and a loxP site in intron 5 of the AML1-gene on chromosome 16 were crossed to mice expressing Cre recombinase. Interchromosomal site-specific recombination at the loxP sites resulted in Cre-mediated chromosomal translocation [t(4;16), t(16;4)] in the offspring. Filled triangles depict loxP sites.

Fig. 2. Targeting of AML1 and ETO in mouse ES cells. The targeting constructs and the genomic regions for the AML1 gene (A) and the ETO gene (B) are shown. Relevant restriction sites and the probes used in Southern blot analysis in (C) and in (D) are presented. loxP sites are depicted as filled triangles flanking the neomycin resistance gene (neo). Exon 5 of AML1 and exon 2 of ETO are presented as white boxes and the direction of transcription is depicted by an arrow. The XbaI fragment size of the wild-type genomic fragment is shown for the AML1 (4601 bp) and ETO (6878 bp) genes. The floxed neomycin gene adds 1836 bp to each fragment. The increased fragment size can be detected in DNA from correctly targeted ES cells for AML1 (C) (6437 bp) and ETO (D) (8714 bp). Positive clones #8, 26, 51 for AML1 and clones #19 and 90 for ETO are shown. bp, base pairs; wt, wild type.

Fig. 3. Cre-mediated chromosomal translocation in primary B cells. A schematic presentation of the pMIG and pMIG–Cre vectors used to generate retroviruses is shown in (A). LTR, long terminal repeat; IRES, internal ribosomal entry site; GFP, green fluorescent protein; Cre, Cre recombinase. Predicted events of Cre-mediated site-specific recombination are presented in (B). Primers a, b, c and d are shown at their position of anealing within the genomic locus as arrows. Neo, neomycin resistance cassette; ex, exon. An agarose gel analyzing the PCRs on DNA isolated from primary B cells is shown in (C). Lanes 1: wild-type cells infected with pMIG–Cre; lanes 2: wild-type cells infected with pMIG; lanes 3: AML^X/ETO^X cells infected with pMIG–Cre; lanes 4: AML^X/ETO^X cells infected with pMIG. Primer pairs used in the PCR are shown above the lanes. M, marker. The band specific for the targeted allele that has recombined out the neomycin cassette is highlighted by a white arrow. RT–PCR analysis on RNA isolated from B cells infected with different retroviruses is shown in (D). Lanes 1: AML^X/ETO^X cells infected with pMIG–Cre; lanes 2: AML^X/ETO^X cells infected with pMIG; lanes 3: wild-type cells infected with pMIG–Cre; lanes 4: wild-type cells infected with pMIG. The upper panel shows the PCR products obtained with primers e and f. The lower panel shows the PCR products obtained with primers e and g. A schematic presentation of the PCRs is shown to the right and the size of the PCR fragment is presented on the left. bp, base pairs.

Fig. 4. Cre-mediated chromosomal translocation in the mouse. Agarose gels analyzing the PCRs on DNA isolated from mouse tails are illustrated in (A). PCR results from mouse #254, 255, 256, 257, 258, 259, 260 and 261 are presented. Schematic presentations of relevant loci and utilized primers (a, b, c and d) are depicted to the right of each panel. Ex, exon. Sequencing reactions obtained from PCR products with primers a + d, and c + d, respectively, are presented in (B). The loxP sites and the respective genomic intron sequences from AML1 and ETO are indicated. PCR analysis on DNA isolated from mouse #260 and indicated tissues is presented in (C). Results with primer pairs c + d, and a + d, respectively are shown. Serial dilution PCR analysis on heart DNA isolated from mouse #260 is presented in (D). Schematic representations of relevant loci and utilized primers (a, b, c and d) are shown on the right.