Predetermined chromosomal deletion encompassing the Nf-1 gene

Abstract

Complex chromosomal rearrangements (deletions, inversions, translocations) are a hallmark of human tumour cells. Yet, the generation of animal models for gross chromosomal abnormalities still presents a formidable challenge. Here, we describe a versatile procedure for chromosomal engineering that was used to generate an ES cell line with a megabase deletion encompassing the tumour suppressor gene neuro®bromatosis-1 (Nf-1) on mouse chromosome 11, which is often deleted in tumours of neural crest origin. Homologous recombination into sites¯anking Nf-1 was used to introduce arti®cial sequences (triple-helix, loxP, vector backbone) that can be employed for in vitro recovery of intervening sequences or the generation of in vivo deletions. This strategy may be developed into a scheme by which large chromosomal regions with precisely de®ned end points may be excised from mammalian cells and reintroduced after suitable in vitro modi®cation.

Engineering chromosomal rearrangements in mice

“…Variations of the aforementioned strategy have been reported by other groups 24,[30][31][32] . Besides pOG231, several other cre-expression vectors, such as pBS185…”

Controlled Somatic and Germline Copy Number Variation in the Mouse Model

“…The phenotypic analysis of the Ts65Dn and Ts1Cje models obtained in a random manner and trisomic for part of the MMU16 [91-93], the Ts1Rhr trisomic for a ~5 Mb Down syndrome critical region (DCR) [72, 94] and the Ts1Yah model trisomic for the syntenic region on Mmu17 [95], both generated by chromosomal engineering, start to reveal the complexity of the genetic interactions at the origin of the disease. Mouse models have also been created for the Smith-Magenis [96-99] and Prader Willi [100] CGS, and to study large genes or cluster of genes such as the HoxD genes cluster [70], the Duchenne muscular dystrophy gene [75], the amyloid precursor protein gene [76] or the Nf1 gene [77]. Cre/loxP chromosome engineering in mouse ES cells was also developed to generate translocations or deletions similar to those found in cancer [86, 101-105].…”

Cre/loxP-Mediated Chromosome Engineering of the Mouse Genome

“…For small regions (less than 100 kb), deletions can be easily selected by cloning, starting from ES cells carrying two loxP sites in cis that are treated with Cre (Zakany and Duboule 1996;Spitz et al 2001). As an alternative, a tk-negative selection marker can be deleted in the rearranged locus (Li et al 1996;Schlake et al 1999;Zhu et al 2000;Nobrega et al 2004). To this end, two targeting vectors that contain the 5 part or the 3 part of the selection cassette with loxP located downstream or upstream, respectively, are inserted consecutively by homologous recombination at the borders of the genomic interval of interest.…”

“…Well-defined chromosomal rearrangements are powerful tools for the functional analysis of the mouse genome to study the function of large genes, including App (Li et al 1996), Xist (Clerc and Avner 1998;Morey et al 2001), Nf1 (Schlake et al 1999), Dkc1 (He et al 2002), TNF/LT (Kuprash et al 2002) and Dystrophin (Kudoh et al 2005). Moreover, they have been used successfully to unravel the relation between function, transcriptional regulation and genomic organisation.…”