Cre-loxP chromosome engineering of a targeted deletion in the mouse corresponding to the 3p21.3 region of homozygous loss in human tumours

Smith, Andrew J.H.; Jian, Xian; Richardson, Melville; Johnstone, Karen A.; Rabbitts, Pamela

doi:10.1038/sj.onc.1205530

Cited by 33 publications

(20 citation statements)

References 21 publications

(26 reference statements)

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“…For one, work in other organisms has shown that CRE is capable of excision and recombination over huge distances [50,12] or even between different chromosomes [13]. In T. brucei, this approach could be used, for example, to delete large tandem gene arrays.…”

Section: Extensions Of Cre/loxpmentioning

confidence: 99%

“…As the enzyme requires no additional co-factors, it has been adapted for genetic manipulation in myriad contexts, including yeast and human cells, in vitro and in vivo [7] (reviewed in [8][9][10]). Most notably, the CRE/loxP system has been used for removing selectable markers [11], excising large tracts of genomic DNA [12], triggering chromosomal rearrangements [13] and integrating exogenous DNA to a specific locus [14], none of which has been achieved in Trypanosoma brucei.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei

Scahill

Pastar

Cross

2008

Molecular and Biochemical Parasitology

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The limited repertoire of drug-resistance markers imposes a serious obstacle to genetic manipulation of Trypanosoma brucei. Here we describe experiments with a fusion protein that allows positive selection for genome integration followed by CRE recombinase-mediated excision of the marker cassette that can be selected by ganciclovir, although the excision event is so efficient that selection is not strictly necessary. We describe two variants of the tetracycline-inducible pLEW100-based CRE-expression vector that reduced its toxicity when stably integrated into the genome, and we demonstrate that transient transfection of circular pLEW100-CRE is highly efficient at catalyzing marker excision. We used this approach to delete the last two enzymes of the pyrimidine synthesis pathway, creating a cell line that is resistant to fluoroorotic acid, which would allow the same enzymes (PYR6-5) to be used as an alternative negative selectable marker.

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Section: Extensions Of Cre/loxpmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei

Scahill

Pastar

Cross

2008

Molecular and Biochemical Parasitology

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“…The first mice lacking RASSF1 were made by Smith et al (88) who generated a targeting vector to the mouse chromosome syntenic to the minimal region of human chromosome 3p21.3 deleted in lung tumors, this 370-kb deletion knocked out 12 genes including rassf1. Heterozygous mice with this contiguous gene deletion were viable and fertile; however, the homozygous null embryos died before 13.5d.p.c.…”

Section: Microtubules Adhesion and Migrationmentioning

confidence: 99%

Role of the Ras-Association Domain Family 1 Tumor Suppressor Gene in Human Cancers

2005

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In recent years, the list of tumor suppressor genes (or candidate TSG) that are inactivated frequently by epigenetic events rather than classic mutation/deletion events has been growing. Unlike mutational inactivation, methylation is reversible and demethylating agents and inhibitors of histone deacetylases are being used in clinical trails. Highly sensitive and quantitative assays have been developed to assess methylation in tumor samples, early lesions, and bodily fluids. Hence, gene silencing by promoter hypermethylation has potential clinical benefits in early cancer diagnosis, prognosis, treatment, and prevention. The hunt for a TSG located at 3p21.3 resulted in the identification of the RAS-association domain family 1, isoform A gene (RASSF1A). RASSF1A falls into the category of genes frequently inactivated by methylation rather than mutational events. This gene is silenced and frequently inactivated by promoter region hypermethylation in many adult and childhood cancers, including lung, breast, kidney, gastric, bladder, neuroblastoma, medulloblastoma, and gliomas. It has homology to a mammalian Ras effector (i.e., Nore1). RASSF1A inhibits tumor growth in both in vitro and in vivo systems, further supporting its role as a TSG. We and others identified the gene in 2000, but already there are over a 150 publications demonstrating RASSF1A methylation in a large number of human cancers. Many laboratories including ours are actively investigating the biology of this novel protein family. Thus far, it has been shown to play important roles in cell cycle regulation, apoptosis, and microtubule stability. This review summarizes our current knowledge on genetic, epigenetic, and functional analysis of RASSF1A tumor suppressor gene and its homologues. Gene IdentificationThe RAS-association domain family 1, isoform A (RASSF1A) tumor suppressor gene (TSG), is a member of a new group of RAS effectors thought to regulate cell proliferation and apoptosis. Formally known as 123F2, two laboratories independently cloned and sequenced RASSF1 (1, 2). Loss of heterozygosity (LOH) studies in lung, breast, and kidney tumors identified several loci in chromosome 3p likely to harbor one or more tumor suppressor genes, including 3p12, 3p14, 3p21.3, and 3p25-26. In 1993, the von HippelLindau (VHL) TSG inactivated in familial and sporadic clear cell renal cell carcinomas was identified from region 3p25 confirming this hypothesis (3). This prompted the search for TSGs within the other regions of 3p. An important TSG was suspected to reside in 3p21.3 because instability of this region is the earliest and most frequently detected deficiency in lung cancer. Overlapping homozygous deletions in lung and breast tumor cell lines reduced the critical region in 3p21.3 to 120 kb and this region was found to be exceptionally gene rich. From this critical region, eight genes were identified, including CACNA2D2 , PL6 /placental protein 6 , CYB561D2/101F6, TUSC4/NPRL2/G21, ZMYND10/BLU, RASSF1/ 123F2, TUSC2/FUS1, and HYAL2/LUCA2. However, des...

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“…These results suggest that ''humanised'' Trp53 mutations have a greater impact on lung tumour progression than complete Trp53 loss [18,19]. Targeting genes deleted early in human lung tumorigenesis, such as the complete cluster at chromosome 3p21.3, showed that heterozygous deletion for this 370 kb region showed no obvious predisposition for lung cancer development albeit homozygous deletion caused embryonal lethality [20]. A more specific deletion of candidate tumour suppressor genes on chromosome 3 like RassF1a, FHIT and VHL, showed that 31% of RassF1a -/-mice produced spontaneous mainly lymphomas but also lung adenomas [21].…”

Section: Transgenic Mice First Generationmentioning

confidence: 90%

Progress and applications of mouse models for human lung cancer

Seranno¹,

Meuwissen²

2010

European Respiratory Journal

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The continued progress of modelling lung cancer in mice has led not only to new means of understanding the molecular pathways governing human lung cancer, but it has also created a vast reservoir of alternative tools to test treatments against this malignancy. More sophisticated somatic mouse models for nonsmall cell lung cancer, small cell lung cancer and pulmonary squamous cell carcinoma have been generated that closely mimic human lung cancer. These models enable us to identify the cells of origin and the role of stem cells in the maintenance of the various types of lung cancer. Moreover, results of lung cancer intervention studies are now starting to reveal the full potential of these somatic mouse models as powerful pre-clinical models.KEYWORDS: Lung cancer, mouse models, nonsmall cell lung cancer, small cell lung cancer P rogress in whole genome approaches to detect genetic alterations in human lung cancer has resulted in the identification of a growing number of lung cancer-related genes. Genome-wide association studies, whether they are based on single-nucleotide polymorphism array studies or detecting changes in gene copy numbers via comparative genomic hybridisation arrays, link the occurrence and frequency of mutations in lung cancer-related genes to the well-defined phenotype of high numbers of human lung cancer. These lung cancer-related genes provide great potential as therapeutic targets for lung cancer intervention. Target validation then occurs through in vitro intervention studies of these specific genetic mutations and their respective molecular pathways in human lung cancer cell lines. However, in vitro cell culture studies are limited and cannot fully mimic the more complex in vivo onset of tumorigenesis and response to tumour therapy. Developing lung cancer in mouse models that harbour specific mutations will undoubtedly provide a further and better insight into the mutation-specific effects on lung tumour biology. Moreover, a high degree of pathophysological similarity between lung tumours from mouse models and their human counterparts will make it possible to use these mouse models for pre-clinical tests. Various intervention strategies against specific mutations can then be tested based on better evaluation of both specificity and efficacy in mouse lung tumours of every developing stage. Continuous innovation of techniques to manipulate the mouse genome has enabled us to adjust compound mouse models of lung cancer in such a way that they start to reproduce the more complex human lung cancer in a higher degree. Complementary to this, the number of genetically engineered mouse models for lung cancer is ever expanding. FIRST MOUSE MODELS FOR LUNG CANCER

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Cre-loxP chromosome engineering of a targeted deletion in the mouse corresponding to the 3p21.3 region of homozygous loss in human tumours

Cited by 33 publications

References 21 publications

CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei

CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei

Role of the Ras-Association Domain Family 1 Tumor Suppressor Gene in Human Cancers

Progress and applications of mouse models for human lung cancer

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