Bloom syndrome (BS) is an autosomal recessive disorder characterized by proportional dwarfism, dysmorphic facial features, photosensitive skin rash, immunodeficiency and the frequent development of a wide variety of malignancies. Cytogenetic studies have shown BS patients to have an increased rate of chromosome breakage and a characteristic elevation of sister chromatid exchange to levels approximately tenfold higher than that seen in normal individuals. Although a link has not been formally established, it is believed that chromosomal instability is responsible for the frequent occurrence of cancer in BS homozygotes. The gene responsible for BS, Blm, has been mapped to 15q26.1 and was recently identified using positional cloning techniques. Blm was found to encode a 1417 amino acid that belongs to the recQ family of DNA helicases. Mutations causing premature termination before the conserved helicase domain within the BLM protein resulted in the BS phenotype. The identification of Blm allows an investigation of the mechanisms by which chromosome breakage and instability might result in the development of malignancy. In order to facilitate such studies, efforts have been initiated to develop a mouse model for BS by targeted disruption of the murine homolog of Blm. Minimally degenerate oligonucleotides based on the sequence of the human version of Blm were used to amplify a PCR product from mouse embryo cDNA. Direct sequencing of this product was consistent with amplification of the murine version of Blm, mBlm. This PCR product was used as a nucleic acid hybridization probe to screen a 129SV mouse genomic DNA phage library. Three clones were identified and the largest of these was subcloned into a plasmid vector to facilitate subsequent analysis. Fluorescence in situ hybridization was used to show that the clone was located on mouse chromosome 7 in the region syntenic to human chromosome 15q26, the band to which hBlm had been mapped. Directed subcloning and sequencing of the genomic fragment was used to determine the location and sequence of seven mBlm exons and the corresponding intron/exon boundaries. This information has been used to design a targeting construct which will be used for disruption of mBlm by homologous recombination in murine embryonic stem cells. The predicted effect of the desired targeting event will be to remove a 119 bp exon within the conserved helicase domain that is believed to be essential for the normal function of Blm. The deletion will cause a frameshift and premature termination of any resulting peptide. This truncation of the BLM peptide is at approximately the same position of the truncation that occurs with the most common mutation found in humans with BS. It is hoped that the generation of this mutation in a mouse homozygous for the disrupted allele will produce a similar phenotype to that seen in patients with Bloom syndrome.
