Functional genomics in mice by tagged sequence mutagenesis

Hicks, Geoffrey; Eg, Shi; Xm, Li; Ch, Li; Pawlak, Maciej R.; He, Renjie

doi:10.1038/ng0897-338

Cited by 112 publications

(84 citation statements)

References 52 publications

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“…The trapped genes were distributed on all chromosomes except the Y, suggesting that a large number of genes across the genome could be accessed by this vector. Some loci seem to be more readily trapped than others, as also found by other large-scale gene trapping efforts using different vectors [19][20][21][22] , and 40 genes were trapped more than once. This phenomenon may be due to recombination hot spots, more permissive integration sites available in larger genes or a limited pool of genes that could be trapped in the genome.…”

Section: Cloning and Analysis Of Trapped Transcriptssupporting

confidence: 68%

“…But all these screens are confined to ES cells or their differentiated derivatives and rely on reporter expression. Sequence-driven screens based on the identity of trapped genes [19][20][21][22]30 are intrinsically biased towards highly annotated genes, and limited functional cues can be deduced from the trapped sequences representing ESTs or new genes. With the gene-trap array, virtually any cell type can now be screened for differentially expressed genes.…”

Section: Discussionmentioning

confidence: 99%

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Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis

et al. 2004

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We developed a versatile, high-throughput genetic screening strategy by coupling gene mutagenesis and expression profiling technologies. Using a retroviral gene-trap vector optimized for efficient mutagenesis and cloning, we randomly disrupted genes in mouse embryonic stem (ES) cells and amplified them to construct a cDNA microarray. With this gene-trap array, we show that transcriptional target genes of platelet-derived growth factor (PDGF) can be efficiently and reliably identified in physiologically relevant cells and are immediately accessible to genetic studies to determine their in vivo roles and relative contributions to PDGF-regulated developmental processes. The same platform can be used to search for genes of specific biological relevance in a broad array of experimental settings, providing a fast track from gene identification to functional validation.

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Section: Cloning and Analysis Of Trapped Transcriptssupporting

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis

et al. 2004

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“…Several smaller-sized mutagenesis screens with gene-trap vectors have been reported (4,(6)(7)(8)(9). However, the use of single gene-trap vectors in each screen, the unavailability of a complete mouse genome sequence, and a comparatively low number of analyzed insertions precluded a systematic assessment of the technology.…”

mentioning

confidence: 99%

A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome

Hansen

Floss²,

Sloun³

et al. 2003

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

141

120

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A major challenge of the postgenomic era is the functional characterization of every single gene within the mammalian genome. In an effort to address this challenge, we assembled a collection of mutations in mouse embryonic stem (ES) cells, which is the largest publicly accessible collection of such mutations to date. Using four different gene-trap vectors, we generated 5,142 sequences adjacent to the gene-trap integration sites (gene-trap sequence tags; http:͞͞genetrap.de) from >11,000 ES cell clones. Although most of the gene-trap vector insertions occurred randomly throughout the genome, we found both vector-independent and vector-specific integration ''hot spots.'' Because >50% of the hot spots were vector-specific, we conclude that the most effective way to saturate the mouse genome with gene-trap insertions is by using a combination of gene-trap vectors. When a random sample of gene-trap integrations was passaged to the germ line, 59% (17 of 29) produced an observable phenotype in transgenic mice, a frequency similar to that achieved by conventional gene targeting. Thus, gene trapping allows a large-scale and cost-effective production of ES cell clones with mutations distributed throughout the genome, a resource likely to accelerate genome annotation and the in vivo modeling of human disease.

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“…DNA flanking the pCX-YNC integration site was cloned from BamHI-digested E10͞YNC genomic DNA by using plasmid rescue as described (19). Genomic DNA captured in the rescued plasmid was sequenced by using the pCX-YNC vector-based primer (TTGTCTCAT-GAGCGGATAC).…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid rescue permits the efficient recovery of flanking DNA to map the insertion site of randomly integrated plasmids (9). We used plasmid rescue to clone a genomic fragment at the junction of the E10͞YNC integration site (19). Sequence from this fragment was used to search the genome for a specific address.…”

Section: An Egfp-based Reporter For Enhancing Genetic Screens In the mentioning

confidence: 99%

Development of an enhanced GFP-based dual-color reporter to facilitate genetic screens for the recovery of mutations in mice

Frank¹,

Meyers²,

Welsh³

et al. 2003

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

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Mutagenesis screens to isolate a variety of alleles leading to null and non-null phenotypes represent an important approach for the characterization of gene function. Genetic schemes that use visible markers permit the efficient recovery of chemically induced mutations. We have developed a universal reporter system to visibly mark chromosomes for genetic screens in the mouse. The dualcolor reporter is based on a single vector that drives the ubiquitous coexpression of the enhanced GFP (EGFP) spectral variants yellow and cyan. We show that widespread expression of the dual-color reporter is readily detected in embryonic stem cells, mice, and throughout developmental stages. CRE-loxP-and FLPe-FRT-mediated deletion of each color cassette demonstrates the modular design of the marker system. Random integration followed by plasmid rescue and sequence-based mapping was used to introduce the marker to a defined genomic location. Thus, single-step placement will simplify the construction of a genomewide bank of marked chromosomes. The dual-color nature of the marker permits complete identification of genetic classes of progeny as embryos or mice in classic regionally directed screens. The design also allows for more efficient and novel schemes, such as marked suppressor screens, in the mouse. The result is a versatile reporter that can be used independently or in combination with the growing sets of deletion and inversion resources to enhance the design and application of a wide variety of genetic schemes for the functional dissection of the mammalian genome.

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Functional genomics in mice by tagged sequence mutagenesis

Cited by 112 publications

References 52 publications

Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis

Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis

A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome

Development of an enhanced GFP-based dual-color reporter to facilitate genetic screens for the recovery of mutations in mice

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