Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutations in vivo.

Gossen, Jan A.; Leeuw, Wiljo J. F. de; Tan, Chung-Wei; Zwarthoff, Ellen C.; Berends, Frits J.; Lohman, P.H.M.; Knook, D.L.; Vijg, Jan

doi:10.1073/pnas.86.20.7971

Cited by 448 publications

(215 citation statements)

References 14 publications

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“…The need for an in vivo assay that can be applied to all organs and tissues led to the development of transgenic reporter based methods. Mice harboring a LacZ or LacI reporter transgene emerged in the late 80s [48–50]. These reporter genes are part of a lambda or plasmid construct that can be recovered from genomic DNA and tested in E. coli for mutational inactivation of the reporter gene.…”

Section: Introductionmentioning

confidence: 99%

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

Gundry

Vijg

2012

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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DNA mutations are the source of genetic variation within populations. The majority of mutations with observable effects are deleterious. In humans mutations in the germ line can cause genetic disease. In somatic cells multiple rounds of mutations and selection lead to cancer. The study of genetic variation has progressed rapidly since the completion of the draft sequence of the human genome. Recent advances in sequencing technology, most importantly the introduction of massively parallel sequencing (MPS), have resulted in more than a hundred-fold reduction in the time and cost required for sequencing nucleic acids. These improvements have greatly expanded the use of sequencing as a practical tool for mutation analysis. While in the past the high cost of sequencing limited mutation analysis to selectable markers or small forward mutation targets assumed to be representative for the genome overall, current platforms allow whole genome sequencing for less than $5,000. This has already given rise to direct estimates of germline mutation rates in multiple organisms including humans by comparing whole genome sequences between parents and offspring. Here we present a brief history of the field of mutation research, with a focus on classical tools for the measurement of mutation rates. We then review MPS, how it is currently applied and the new insight into human and animal mutation frequencies and spectra that has been obtained from whole genome sequencing. While great progress has been made, we note that the single most important limitation of current MPS approaches for mutation analysis is the inability to address low-abundance mutations that turn somatic tissues into mosaics of cells. Such mutations are at the basis of intra-tumor heterogeneity, with important implications for clinical diagnosis, and could also contribute to somatic diseases other than cancer, including aging. Some possible approaches to gain access to low-abundance mutations are discussed, with a brief overview of new sequencing platforms that are currently waiting in the wings to advance this exploding field even further.

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Section: Introductionmentioning

confidence: 99%

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

Gundry

Vijg

2012

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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“…Extrapolation from animal data has been used in an effort to understand IRinduced germline mutations in humans due to the inherent difficulties in human epidemiological studies involving germ cells [1]. Several rodent assays are capable of detecting germline mutation, including the specific locus (SL) test [2], heritable translocation (HT) assay [3], dominant lethal (DL) assay [4], expanded simple tandem repeats (ESTRs) assay [5], and transgenic rodent assays [6,7]. The traditional mutation assays, e.g.…”

Section: Introductionmentioning

confidence: 99%

Recovery of a low mutant frequency after ionizing radiation-induced mutagenesis during spermatogenesis

Intano

McCarrey

et al. 2008

Mutation Research/Genetic Toxicology and Environmental Mutagene

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Humans are exposed to ionizing radiation (IR) under various circumstances, e.g. cosmic radiation, diagnostic X-rays and radiotherapy for cancer. It has been shown that IR can impair spermatogenesis and can cause mutations in germ cells. However, the mutagenic responses of germ cells exposed to IR at different stages of testicular maturation have not been examined by directly assessing the mutant frequency in defined spermatogenic cell types. This study was performed to address whether preadult exposure to IR can increase mutations in adult germ cells that could in turn have a major impact on adult reproductive function and the health of ensuing offspring. Male Lac I transgenic mice were irradiated with a single dose of 2.5 Gy of γ-ray at different ages before adulthood, reflecting different stages of testicular maturation, and then mutant frequency (MF) was determined directly in spermatogenic cell types emanating from the irradiated precursor cells. The results showed that (1) preadult exposure to IR did not significantly increase MF in adult epididymal spermatozoa; (2) spermatogenic stages immediately following the irradiated stage(s) displayed an elevated mutant frequency, but (3) the mutant frequency was restored to unirradiated levels in later stages of spermatogenesis. These findings provide evidence that there is a mechanism(s) to prevent spermatogenic cells with elevated mutant frequencies from progressing through spermatogenesis.

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“…The pUR288 transgene harbors a shuttle vector with a lacZ reporter gene to examine mutagenesis in vivo with greater ease than studying endogenous genes (Boerrigter et al, 1995). The plasmid-based pUR288 vector also permits, in contrast to previously developed shuttle vectors based on phage l (Gossen et al, 1989;Kohler et al, 1991), the detection of a broad range of recombination mutations, including rearrangements of the reporter gene with mouse genomic sequences and large deletions within the reporter gene. The combined features of the l-MYC and pUR288 transgenes rendered l-MYC/pUR288 mice uniquely valuable for assessing MYC-induced genomic instability in a mature B-cell lymphoma (mouse BL).…”

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confidence: 99%

Genomic instability in mouse Burkitt lymphoma is dominated by illegitimate genetic recombinations, not point mutations

Rockwood

Torrey²,

Kim

et al. 2002

Oncogene

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l-MYC-induced mouse Burkitt lymphoma (BL) harboring the shuttle vector pUR288, which includes a lacZ reporter gene to study mutagenesis, was employed to assess genomic instability associated with MYC deregulation. The frequency of lacZ mutations in lymphomas was elevated only 1.75-fold above that in normal tissue, indicating that mouse BL does not exhibit a phenotype of hypermutability. However, the nature of lacZ mutations was strikingly different in normal tissues and lymphomas. While point mutations comprised approximately 75% of the mutations found in normal tissues, apparent translocations, deletions and inversions constituted the majority of mutations (*65%) in lymphomas. Genomic instability in mouse BL thus seems characterized by a preponderance of illegitimate genetic rearrangements in the context of near-background mutant frequencies. SKY analyses of cell lines from primary BL tumors revealed substantial changes in chromosomal structure, confirming the lacZ studies. Bi-allelic deletions of the tumor suppressor p16 Ink4a were detected in six out of 16 cell lines, illustrating cellular selection of advantageous mutations. Together, these approaches indicate that MYC may contribute to lymphomagenesis through the dominant mutator effect of inducing chromosomal instability. The results further suggest that a phenotype of hypermutability (elevated mutant frequency) may not always be required for oncogenesis to occur.

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Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutations in vivo.

Cited by 448 publications

References 14 publications

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

Recovery of a low mutant frequency after ionizing radiation-induced mutagenesis during spermatogenesis

Genomic instability in mouse Burkitt lymphoma is dominated by illegitimate genetic recombinations, not point mutations

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