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.

Review: genetic models of acute myeloid leukaemia

“…The tetracycline (tet-) inducible (Gossen and Bujard, 1992;Gossen et al, 1995;Jaisser, 2000;Ryding et al, 2001) and Cre/loxPmediated (Muller, 1999;Jaisser, 2000;Testa and Stewart, 2000;Ryding et al, 2001;Yu and Bradley, 2001) interchromosomal translocation recombination systems have been utilized in generation of inducible models of AML1/ETO t(8;21) (Buchholz et al, 2000;Higuchi et al, 2002;Rhoades et al, 2000), MLL-CBP t(11;16) , MN1-TEL t(12 ;22) (Kawagoe and Grosveld, 2005) and MLL/AF9 t(9;11) (Collins et al, 2000). In both the tet-off and Cre-loxP models of AML1/ETO (Rhoades et al, 2000;Higuchi et al, 2002), the transcriptional control of AML1/ETO fusion protein was highly expressed in bone marrow, however, as noted in the hMRP8-AML1/ETO model, there was no development of leukaemic phenotype unless expressed with cooperating mutations.…”

A conditional model of MLL-AF4 B-cell tumourigenesis using invertor technology

“…The entire fragment from the clone (AF4-pA-MC1Neo-pA) was excised as an Sfi1 fragment, the restriction site ends filled in and the fragment was introduced into the blunted BamHI site of pLOXKI so that the AF4-pA-MC1Neo-pA sequences were immediately downstream of the short intron sequence and acceptor splice site in pLOXKI and all flanked by loxP sites (to give pLOXKI-AF4). The invertor cassette of pLOXKI-AF4 was cloned as a SceI-ClaI blunt fragment into the blunt-ended BglII site of the Mlltargeting vector (a 5.5 kb EcoRI fragment (Collins et al, 2000)). Finally the Herpes simplex virus (HSV) tk gene was added as a blunt end filled XbaI fragment into the blunted ClaI site of the Mll-targeting vector.…”

“…Filter hybridization of SphI-digested genomic DNA from various tissues was carried out using the Mll 5 0 probe (Collins et al, 2000) (Figure 1a) to detect the inverted allele in the various organs. To estimate the proportion of inverted vs noninverted Mll-AF4 alleles in various cell populations, DNA was prepared (Qiagen Blood Kit, Qiagen, Hilden, Germany) from 10 000 unsorted or FACS-sorted cells expressing Mac-1, B220 or Thy1.2 Purity of the sorted populations was X97.5%.…”

Leukaemia lineage specification caused by cell-specific Mll-Enl translocations

“…These questions have been explored further by generating different mouse lines where conditional de novo Mll-Enl or Mll-Af9 reciprocal chromosomal translocations occur (translocator mice; Collins et al, 2000;Forster et al, 2003) using Cre-mediated inter-chromosomal recombination. Different lineage-specific Cre genes have been employed to achieve cell-specific translocations and we have found that all the translocator mice involving Mll-Enl fusion, except those where the Cre recombinase was expressed from the CD19 gene, developed leukaemia.…”