Mouse Models Generated By Chromosome Engineering

During the last decade, technology advances have already made generating large chromosome rearrangements possible in the mouse. Continuous development of new technologies will undoubtedly further facilitate our ability to engineer many kinds of chromosome changes associated with human disorders. Some of the recent successes that utilized chromosome engineering to model genomic disorders and to genetically dissect human disease are briefly described.

Fig. 3. Construction of targeting vectors by recombineering. (A) A bacterial artificial chromosome (BAC) with an approx 150-kb genomic DNA insert is transformed to recombineering competent Escherichia coli cells (DY380). The recombination substrate, 5'HPRT cassette, together with a Neo/Kan cassette, can be recombined using short homology arms (200-500 bp) to a precise position in the BAC with recombineering. (B) In the next step, a retrieval vector, which has two short homology arms (200500 bp) corresponding to the ends of the interested genomic region, is linearized and electroporated to the BAC containing DY380 cells. (C) Homologous recombination (gap repair) between the two homology arms on the retrieval vector and the sequences on the BAC results in cloning of the genomic DNA and the targeted 5' HPRT-Neo cassette from the BAC to the retrieval vector. Different genetic elements such as a TK cassette (Herpes simplex virus thymidine kinase gene) for negative selection in embryonic stem cells can be engineered to the retrieval vector. A detailed description of this approach can be found in ref. 49. pSK, plasmid pBluescript SK.

Fig. 3. Construction of targeting vectors by recombineering. (A) A bacterial artificial chromosome (BAC) with an approx 150-kb genomic DNA insert is transformed to recombineering competent Escherichia coli cells (DY380). The recombination substrate, 5'HPRT cassette, together with a Neo/Kan cassette, can be recombined using short homology arms (200-500 bp) to a precise position in the BAC with recombineering. (B) In the next step, a retrieval vector, which has two short homology arms (200500 bp) corresponding to the ends of the interested genomic region, is linearized and electroporated to the BAC containing DY380 cells. (C) Homologous recombination (gap repair) between the two homology arms on the retrieval vector and the sequences on the BAC results in cloning of the genomic DNA and the targeted 5' HPRT-Neo cassette from the BAC to the retrieval vector. Different genetic elements such as a TK cassette (Herpes simplex virus thymidine kinase gene) for negative selection in embryonic stem cells can be engineered to the retrieval vector. A detailed description of this approach can be found in ref. 49. pSK, plasmid pBluescript SK.

del22q11.2 Syndrome del22qll.2 syndrome (also known as DiGeorge syndrome [DGS], velocardiofacial syndrome [VCFS], and conotruncal anomaly face syndrome) with an incidence of 1 in every 4000-5000 births, is the most frequent chromosomal microdeletion syndrome known (52). The common deletion is caused by LCR-mediated recombination (53-55). Typical patient phenotype includes cardiovascular defects, thymic, parathyroid, craniofacial anomalies, and learning disabilities. Many of the features (e.g., heart outflow tract, craniofacial, velopharyn-

geal, ear, thymic, and parathyroid abnormalities) are attributable to developmental defects of the embryonic pharyngeal apparatus.

Although at least 30 genes have been mapped to the common deletion region of del22qll.2, until recently, none of these genes had been shown to carry genetic mutations in the patients who do not have the common deletion. The mouse syntenic region of the del22q11.2 genomic region is located on the mouse chromosome 16. Using chromosome engineering, a 1.2-Mb deletion (Es2-Ufd1l) was generated and the deletion was subsequently established in the mouse germline (56). Some of the embryos and adult mice carrying the deletion in heterozygous status manifested cardiovascular defects on a mixed genetic background. However, these deletion mice did not have other phenotypes associated with the 22q11.2 deletion in the human patients. Nevertheless, the mutant mice did recapitulate one important phenotype of the del22q11.2 syndrome, confirming that the disease-causing gene(s) is within this deletion interval.

To narrow the critical genomic region, Lindsay et al. (57) generated several smaller deletions and found that one deletion containing the transcription factor gene, Tbx1, is responsible for the cardiovascular defects in the mouse. Two additional evidences supported Tbx1 is the causal gene. First, a P1 artificial chromosome containing Tbx1 rescued the cardiovascular defects in the deltion mice. Second, a Tbx1 hypomorphic allele confirmed that Tbx1 is a dosage sensitive gene in the mouse and is required for normal development of the pharyngeal arch arteries (57). In an independent study, a chromosomal deletion encompassing the Tbx1 locus was generated by crossing two loxP sites, that were in the del22q11.2 critical region but located on the two homologous chromosome 16, to cis configuration (58). The deletion generated by Cre-loxP spanned approx 1 Mb and the heterozygous deletion mice had similar cardiovascular defects as found in the mice engineered by Lindsay et al. (57).

To further confirm the role of TBX1 in del22q11.2, a null allele of Tbx1 was generated by gene targeting. Interestingly, although mice heterozygous for this null allele of Tbx1 only had cardiac outflow tract anomalies (identical to the phenotype in the deletion heterozygotes), the Tbx1 -/- mice displayed a wide range of developmental anomalies encompassing almost all of the common DGS/VCFS features, including hypoplasia of the thymus and parathyroid glands, cardiac outflow tract abnormalities, abnormal facial structures, abnormal vertebrae, and cleft palate (59). Taken these data together, analysis of the genetically engineered mice strongly supported that haploinsufficiency of TBX1 is responsible for most clinical findings in del22q11.2 patients. This hypothesis has been confirmed by recent studies that identified TBX1 mutations in patients with a typical del22q11.2 phenotype but with no apparent genomic deletions (60).

The Tbx1 analyses represent an excellent demonstration of the power of using genetically modified mice to dissect the molecular basis of a human disease. It is interesting to note that the Tbx1 gene appears to be more sensitive to dosage reduction in human than in the mouse. This may reflect species difference, an important aspect that should be taken into account when interpreting data obtained from genetically modified mouse models. It has been postulated that humans are more sensitive than the mouse to lower levels of the dosage-sensitive gene products and, thus, display increased phenotype penetrance (59). In support of this view, in several other cases, including MSX2 and PAX9, heterozygous effects are evident in humans but not in the mouse, but the homozygous effects in mouse are similar in nature to the human heterozygous effects (61-64). Another important lesson learned from genetic dissection of del22q11.2 in the mouse is that genetic heterogeneity may be common for human disease. Analyses of hundreds of patients with apparent del22q11.2 phenotype but without the deletion failed to identify any TBX1 mutations in several previous studies (57,58). Identifying the TBX1 mutations became possible only when the studies were implemented in the potential mutation-carrying patients with extremely careful clinical examination (60).

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