X-ray-induced mutations in mouse embryonic stem cells

Thomas, James W.; LaMantia, Christian; Magnuson, Terry

doi:10.1073/pnas.95.3.1114

Cited by 61 publications

(41 citation statements)

References 48 publications

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“…This result underscores the tight control of the euploid ES cell genome. A region of haploinsufficiency has also been proposed to reside on Chr 9 in studies on a radiation-induced deletion complex (19). The observation of haploinsufficiency in ES cells is in direct contrast with many cancer cells that often carry large chromosomal deletions and chromosomal losses.…”

Section: Discussionmentioning

confidence: 99%

Engineering Mouse Chromosomes with Cre-loxP: Range, Efficiency, and Somatic Applications

Zheng¹,

Sage²,

Sheppeard³

et al. 2000

Molecular and Cellular Biology

193

137

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Chromosomal rearrangements are important resources for genetic studies. Recently, a Cre-loxP-based method to introduce defined chromosomal rearrangements (deletions, duplications, and inversions) into the mouse genome (chromosome engineering) has been established. To explore the limits of this technology systematically, we have evaluated this strategy on mouse chromosome 11. Although the efficiency of Cre-loxPmediated recombination decreases with increasing genetic distance when the two endpoints are on the same chromosome, the efficiency is not limiting even when the genetic distance is maximized. Rearrangements encompassing up to three quarters of chromosome 11 have been constructed in mouse embryonic stem (ES) cells. While larger deletions may lead to ES cell lethality, smaller deletions can be produced very efficiently both in ES cells and in vivo in a tissue-or cell-type-specific manner. We conclude that any chromosomal rearrangement can be made in ES cells with the Cre-loxP strategy provided that it does not affect cell viability. In vivo chromosome engineering can be potentially used to achieve somatic losses of heterozygosity in creating mouse models of human cancers.Specific chromosomal rearrangements can be engineered in mice to model human chromosomal disorders, such as those associated with deletions or duplications of chromosomal segments (for example, Smith-Magenis syndrome, Downs syndrome, and Charcot-Marie-Tooth type 1A) (5, 7, 10). Chromosomal rearrangements also facilitate genetic studies (2, 14). Inversion chromosomes can be used to establish balanced lethal systems to facilitate stock maintenance. Deletions can be used for mapping and in genetic screens for recessive mutations.In Drosophila melanogaster there is a wealth of chromosomal rearrangements that are widely used as genetic tools. In particular, chromosomal deletions (deficiencies) which collectively cover approximately 60 to 70% of the genome have been indispensable in mapping recessive mutations and in regionspecific mutagenesis screens. The use of deletions in mice, however, has been much more limited because of the paucity of chromosomal deletions which, until recently, were restricted to a few regions of the mouse genome flanking visible genetic markers (14). The application of the Cre-loxP recombination system over large distances in mouse embryonic stem (ES) cells has made it possible to engineer specific chromosomal rearrangements in the mouse (13,17). This chromosome engineering strategy involves three manipulation steps in ES cells (see Fig. 1): (i) one loxP site is targeted to one endpoint along with the 5Ј half of an Hprt selectable marker gene (5Ј hprt); (ii) another loxP site is targeted to a second endpoint with the 3Ј half of the Hprt gene (3Ј hprt); and (iii) transient expression of Cre recombinase catalyzes loxP site-specific recombination, leading to the desired rearrangement. Reconstitution of a fulllength Hprt gene provides selection for ES cells with the recombination products in culture in HAT (hypoxanthine-amin...

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Section: Discussionmentioning

confidence: 99%

Engineering Mouse Chromosomes with Cre-loxP: Range, Efficiency, and Somatic Applications

Zheng¹,

Sage²,

Sheppeard³

et al. 2000

Molecular and Cellular Biology

193

137

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“…This is especially true for small genomic regions (such as the human 11p14-p15 homology region discussed here) where mutation ordering with respect to deletion breakpoints greatly facilitates the selection and analysis of candidate genes. New techniques of creating heritable deletions in embryonic stem cells, either by Cre-loxP-mediated chromosome engineering (38)(39)(40) or by radiation and negative selection (41)(42)(43)(44)(45), will allow the continued development of fine-structure point-mutation maps in other mutagenized regions of the genome that can then be correlated easily with DNA-sequence and transcription maps rapidly emerging from human and mouse genome projects. In each case, females heterozygous for the Del(p) deletions shown [p x ͞Del(p)] were crossed to an obligate carrier (progeny-tested) ru2 m p͞ru2 ϩ ϩ male, with m representing a mutation at the indicated locus.…”

Section: Discussionmentioning

confidence: 99%

Functional annotation of mammalian genomic DNA sequence by chemical mutagenesis: A fine-structure genetic mutation map of a 1- to 2-cM segment of mouse chromosome 7 corresponding to human chromosome 11p14-p15

Rinchik

Carpenter

Johnson

2002

Proc. Natl. Acad. Sci. U.S.A.

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Eleven independent, recessive, N-ethyl-N-nitrosourea-induced mutations that map to a Ϸ1-to 2-cM region of mouse chromosome (Chr) 7 homologous to human Chr 11p14-p15 were recovered from a screen of 1,218 gametes. These mutations were initially identified in a hemizygous state opposite a large p-locus deletion and subsequently were mapped to finer genomic intervals by crosses to a panel of smaller p deletions. The 11 mutations also were classified into seven complementation groups by pairwise crosses. Four complementation groups were defined by seven prenatally lethal mutations, including a group (l7R3) comprised of two alleles of obvious differing severity. Two allelic mutations (at the psrt locus) result in a severe seizure and runting syndrome, but one mutation (at the fit2 locus) results in a more benign runting phenotype. This experiment has added seven loci, defined by phenotypes of presumed point mutations, to the genetic map of a small (1-2 cM) region of mouse Chr 7 and will facilitate the task of functional annotation of DNA sequence and transcription maps both in the mouse and the corresponding human 11p14-p15 homology region.N-ethyl-N-nitrosourea ͉ regional mutagenesis ͉ allelic series ͉ seizures and runting ͉ prenatal and juvenile abnormalities T he supermutagenicity of N-ethyl-N-nitrosourea (ENU) (1) has made it possible to recover heritable mutations at high frequency from mouse spermatogonial stem cells. Consequently, it is possible to use phenotype-driven strategies to recover new ENU-induced mutations in specific, preselected loci for the generation of new alleles (1-4), in whole-genome screens for genes comprising components of complex pathways or phenotypes (5-10), or, with the use of chromosomal rearrangements, in a wide variety of genes within specific chromosomal regions (11-18). The phenotyping protocols used in such strategies can range from simple to complex and can be narrowly or broadly focused, with the output of any experiment dependent largely on the discriminating power of the phenotyping used. The extensive homologies between the genomes of mouse and human make it feasible to use the mouse for discovery of new mutations and phenotypes that aid in the functional annotation of the corresponding human DNA sequence and transcription maps.The region of mouse chromosome (Chr) 7 surrounding the pink-eyed dilution (p) locus that shares homology with human genomic regions 15q11-q13 and 11p14-p15 is being characterized by both genetic and molecular approaches (15,(19)(20)(21)(22)(23)(24)(25)(26). We previously reported initial results of a hemizygosity-screen strategy for recovering ENU-induced recessive mutations within the 4-to 5-cM Del(ru2 p) 46DFiOD deletion (15); this strategy is similar to a mutation-recovery experiment carried out for a 6-to 11-cM deletion at the more distally mapping Chr 7 tyrosinase [Tyr; previously, albino (c)] locus (14,16). The initial phase of this ''p-region screen'' (like the entire Tyr-region screen) used simple phenotyping criteria such as recognizing lethal...

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“…Evidence for haploinsufficient loci on chromosome 9 in ES cells also was reported by Thomas et al, who created a radiation-induced deletion complex at the Ncam locus (Thomas et al 1998), and Zheng et al, who induced deletions on chromosome 11 with a targeted Cre-loxP strategy (Zheng et al 2000).…”

Section: Discussionmentioning

confidence: 82%

“…To create a series of deletions spanning the proximal region of mouse chromosome 5, the technique of irradiating F 1 hybrid ES cells was used (You et al 1997a(You et al , 1997bThomas et al 1998). This strategy entails the targeted insertion of a tk-expressing cassette into a locus of choice, treatment of targeted cells with radiation, and selection for loss of tk expression with the drug FIAU (1-2'-deoxy-2Ј-fluoro-␤-D-arabinofuranosyl-5-iodouracil).…”

Section: Resultsmentioning

confidence: 99%

Interdigitated Deletion Complexes on Mouse Chromosome 5 Induced by Irradiation of Embryonic Stem Cells

Schimenti¹,

Libby²,

Bergstrom³

et al. 2000

Genome Res.

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Chromosome deletions have several applications in the genetic analysis of complex organisms. They can be used as reagents in region-directed mutagenesis, for mapping of simple or complex traits, or to identify biological consequences of segmental haploidy, the latter being relevant to human contiguous gene syndromes and imprinting. We have generated three deletion complexes in ES (Embryonic Stem) cells that collectively span ∼ 40 cM of proximal mouse chromosome 5. The deletion complexes were produced by irradiation of F 1 hybrid ES cells containing herpes simplex virus thymidine kinase genes (tk) integrated at the Dpp6, Hdh (Huntington disease locus), or Gabrb1 loci, followed by selection for tk-deficient clones. Deletions centered at the adjacent Hdh and Dpp6 loci ranged up to ∼ 20 cM or more in length and overlapped in an interdigitated fashion. However, the interval between Hdh and Gabrb1 appeared to contain a locus haploinsufficient for ES cell viability, thereby preventing deletions of either complex from overlapping. In some cases, the deletions resolved the order of markers that were previously genetically inseparable. A subset of the ES cell-bearing deletions was injected into blastocysts to generate germline chimeras and establish lines of mice segregating the deletion chromosomes. At least 11 of the 26 lines injected were capable of producing germline chimeras. In general, those that failed to undergo germline transmission bore deletions larger than the germline-competent clones, suggesting that certain regions of chromosome 5 contain haploinsufficient developmental genes, and/or that overall embryonic viability is cumulatively decreased as more genes are rendered hemizygous. Mice bearing deletions presumably spanning the semidominant hammertoe locus (Hm) had no phenotype, suggesting that the classic allele is a dominant, gain-of-function mutation. Overlapping deletion complexes generated in the fashion described in this report will be useful as multipurpose genetic tools and in systematic functional mapping of the mouse genome.

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X-ray-induced mutations in mouse embryonic stem cells

Abstract: Deletion complexes consisting of multiple chromosomal deletions induced at single loci can provide a means for functional analysis of regions spanning several centimorgans in model genetic systems. A strategy to identify and map deletions at any cloned locus in the mouse is described here.

Cited by 61 publications

References 48 publications

Engineering Mouse Chromosomes with Cre-loxP: Range, Efficiency, and Somatic Applications

Engineering Mouse Chromosomes with Cre-loxP: Range, Efficiency, and Somatic Applications

Functional annotation of mammalian genomic DNA sequence by chemical mutagenesis: A fine-structure genetic mutation map of a 1- to 2-cM segment of mouse chromosome 7 corresponding to human chromosome 11p14-p15

Interdigitated Deletion Complexes on Mouse Chromosome 5 Induced by Irradiation of Embryonic Stem Cells

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