Targeted oncogene activation by site-specific recombination in transgenic mice.

Lakso, Merja; Sauer, Brian; Mosinger, B; Lee, E J; Manning, Robert; Yu, Shuhua; Mulder, K L; Westphal, Heiner

doi:10.1073/pnas.89.14.6232

Cited by 616 publications

(397 citation statements)

References 27 publications

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“…We generated the R172P substitution (CGC to CCC) at nucleotide 515 (taking the A in the first ATG as nucleotide number 1) in exon 5 of Trp53 by site-directed mutagenesis. We crossed mice with germline transmission of the targeted allele to CMV-cre transgenic mice 29 to effect germline deletion of the PGK-neo r cassette. We crossed the resulting mice with C57BL/6 mice to generate Trp53 515C/515C mice.…”

Section: Methodsmentioning

confidence: 99%

Chromosome stability, in the absence of apoptosis, is critical for suppression of tumorigenesis in Trp53 mutant mice

et al. 2003

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The p53 protein integrates multiple upstream signals and functions as a tumor suppressor by activating distinct downstream genes 1-3 . At the cellular level, p53 induces apoptosis, cell cycle arrest and senescence. A rare mutant form of p53 with the amino acid substitution R175P, found in human tumors, is completely defective in initiating apoptosis but still induces cell cycle arrest 4,5 . To decipher the functional importance of these pathways in spontaneous tumorigenesis, we used homologous recombination to generate mice with mutant p53-R172P (the mouse equivalent of R175P in humans). Mice inheriting two copies of this mutation (Trp53 515C/515C ) escape the early onset of thymic lymphomas that characterize Trp53-null mice. At 7 months of age, 90% of Trp53-null mice had died, but 85% of Trp53 515C/515C mice were alive and tumor-free, indicating that p53-dependent apoptosis was not required for suppression of early onset of spontaneous tumors. The lymphomas and sarcomas that eventually developed in Trp53 515C/515C mice retained a diploid chromosome number, in sharp contrast to aneuploidy observed in tumors and cells from Trp53-null mice. The ability of mutant p53-R172P to induce a partial cell cycle arrest and retain chromosome stability are crucial for suppression of early onset tumorigenesis.We introduced the 515G→C mutation into the Trp53 locus by homologous recombination into embryonic stem (ES) cells and cre-loxP-mediated excision in mice (Fig. 1). Sequencing of the entire coding region of the Trp53 transcript from a homozygous mutant mouse found no other changes. The allele Trp53 515C expressed a fulllength mutant p53 protein with the amino acid substitution R172P, which was more abundant than wild-type p53 (Fig. 1d).To assay the cell cycle arrest function, we treated subconfluent cultures of wild-type, Trp53-null, and Trp53 515C/515C mouse embryonic fibroblasts (MEFs) with γ-radiation and labeled them with 5-bromo-deoxyuridine (BrdU) to measure the number of cells in S phase 6 . The ratio of cells in S phase among cells treated with γ-radiation versus untreated cells was significantly lower in Trp53 515C/515C MEFs than in Trp53-null MEFs (Fig. 2a), but the

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Section: Methodsmentioning

confidence: 99%

Chromosome stability, in the absence of apoptosis, is critical for suppression of tumorigenesis in Trp53 mutant mice

et al. 2003

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“…After establishment of a transgenic line, the STOP signal can be removed by Cre recombinase-mediated excision to activate the transgene expression. 5 Thus a properly designed targeting construct containing loxP sites can be used for temporally or spatially controlled knockout or for introducing subtle mutations (for a review of the control of transgenes, see Sauer 6 ).…”

Section: Introductionmentioning

confidence: 99%

“…5 The SV40 tumor antigen was rendered completely quiescent by the insertion of a synthetic STOP sequence between it and a lens-specific promoter. The resulting transgenic lines exhibited no incidence of tumor formation.…”

Section: Introductionmentioning

confidence: 99%

MicroPET imaging of Cre–loxP-mediated conditional activation of a herpes simplex virus type 1 thymidine kinase reporter gene

et al. 2004

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“…The targeting vector used had the implicated GAG removed in exon 5 of Dyt1 and a STOP sequence (Lakso et al, 1992) containing a false translation signal, splice donor site and a poly(A) tail inserted into intron 4 (Fig. 1a).…”

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confidence: 99%

Motor deficits and hyperactivity in Dyt1 knockdown mice

Dang

Yokoi²,

Pence³

et al. 2006

Neuroscience Research

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The DYT1 gene containing a trinucleotide deletion (ΔGAG) is linked to early-onset dystonia, a neurological movement disorder of involuntary muscle contractions. To understand Dyt1's contribution to dystonia, we produced and analyzed Dyt1 knockdown (KD) mice that expressed a reduced level of torsinA protein encoded by Dyt1. Knockdown mice exhibited deficits in motor control and a decreased trend in dopamine with a significant reduction in 3,4-dihydroxyphenylacetic acid. These alterations are similar to those displayed by previously reported Dyt1 ΔGAG knockin heterozygous mice, suggesting that the partial loss of torsinA function contributes to the pathology of the disease. KeywordsDyt1; torsinA; dystonia; knockdown; Tor1A; Dyt1 ΔGAG A trinucleotide deletion (ΔGAG) in the DYT1 gene is found in a majority of patients with Oppenheim's early-onset dystonia, a neurological disorder of uncontrollable muscle contractions (Ozelius et al., 1997). DYT1 codes for torsinA protein, and the ΔGAG deletion removes a glutamic acid (ΔE) from the protein. The role of mutant torsinA in the development of dystonia is unknown, but possible functions of normal torsinA were reported to include its involvement in cytoskeletal dynamics, nuclear membrane formation, and neuroprotection (Kuner et al., 2003;Bragg et al., 2004;Gonzalez-Alegre & Paulson, 2004;Goodchild & Dauer, 2004;Shashidharan et al., 2004;Hewett et al., 2006;Kock et al., 2006). Also unknown is the nature of the genetic mutation in torsinA, an important aspect of the pathophysiology of dystonia that may affect the development of genetic-based therapeutics.The ΔGAG mutation of DYT1 has been speculated to work through a toxic-gain-of-function mechanism by reports of protein aggregates caused by an overexpression of mutant torsinA in cultured cells and Drosophila (Hewett et al., 2000;Kustedjo et al., 2000;Koh et al., 2004). Also, in patient tissues and two mouse models, an overexpression transgenic and our Dyt1 ΔGAG knockin (Dyt1 ΔGAG) mouse lines, aggregates containing torsinA and ubiquitin were found primarily in the brainstem, even though torsinA is widely expressed throughout the brain Dang et al., 2005;Shashidharan et al., 2005). In contrast, other published findings have suggested that ΔGAG causes a loss of normal protein function (Torres et al., 2004;Goodchild et al., 2005). For example, nuclear envelope abnormalities were noted * To whom correspondence and proofs should be addressed. Yuqing Li, 3347 Beckman Institute, 405 N. Mathews Ave., Urbana, Illinois, 61801, Tel.:217-333-4002, Fax: 217-244-1726, E-mail: y-li4@uiuc.edu.. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all l...

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Targeted oncogene activation by site-specific recombination in transgenic mice.

Cited by 616 publications

References 27 publications

Chromosome stability, in the absence of apoptosis, is critical for suppression of tumorigenesis in Trp53 mutant mice

Chromosome stability, in the absence of apoptosis, is critical for suppression of tumorigenesis in Trp53 mutant mice

MicroPET imaging of Cre–loxP-mediated conditional activation of a herpes simplex virus type 1 thymidine kinase reporter gene

Motor deficits and hyperactivity in Dyt1 knockdown mice

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