The Centre for Modeling Human Disease Gene Trap resource

To, Christine; Epp, Trevor; Reid, Tammy; Lan, Qing; Yu, Man; Li, Carol Y. J.; Ohishi, Minako; Hant, Paula; Tsao, Nora; Casallo, Guillermo; Rossant, Janet; Osborne, Lucy R.; Stanford, William

doi:10.1093/nar/gkh106

Cited by 28 publications

(17 citation statements)

References 16 publications

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“…When the same clones of Venus-positive lines were compared between the undifferentiated and the EB cultures for both pGTIV1 and pGTIV2, up to 30% of the lines demonstrated differential expression pattern of Venus after the induction of differentiation. These results are comparable or even better than similar gene-trap vectors utilizing the standard histochemical b-gal reporter developed by and used by our lab (Table 1; http:// www.cmhd.ca/genetrap/index.html; To et al, 2004).…”

supporting

confidence: 58%

Development of a gene‐trap vector with a highly sensitive fluorescent protein reporter system for expression profiling

Tanaka

Davey

Lan

et al. 2008

Genesis

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Combining high-content screening (HCS) with random gene-trap mutagenesis could be a powerful tool to investigate transcriptional networks, cell signaling, chemical genetics, and developmental processes. However, a critical limitation has been poor quantification of reporter expression per cell. To overcome this hurdle, we generated a variety of Gtx-based expression cassettes and re-evaluated translational enhancement of arrayed Gtx segments in tandem by HCS. We then modified the cassette into a new polyA trap vector, which consists of a variant of yellow fluorescent protein, Venus, in combination with the Gtx segments. Expression of Venus was detected in about 60% of trapped genes assayed in embryonic stem cell (ESC) cultures, comparable to expression screening of LacZ-based vectors. Furthermore, tetraploid aggregations using a clone encoding a gene-trap insertion into Twist2 demonstrated identical spatiotemporal expression between Venus and Twist2. This highly sensitive reporter system is amenable to high-throughput expression-based real-time HCS including single cell analyses.

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supporting

confidence: 58%

Development of a gene‐trap vector with a highly sensitive fluorescent protein reporter system for expression profiling

Tanaka

Davey

Lan

et al. 2008

Genesis

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“…This resource, which includes gene-trap clones specific for endothelial cells as well as other cell types, can be searched by both sequence and expression patterns at http://www.cmhd.ca/sub/ genetrap.asp. 48 By using this new generation of gene-trap vectors, the gene-trap screening strategy described here can be useful to identify endothelial-specific genes and to analyze their function in mice.…”

Section: Discussionmentioning

confidence: 99%

Gene-trap expression screening to identify endothelial-specific genes

Hirashima

Bernstein²,

Stanford³

et al. 2004

Blood

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The endothelial cell is a key cellular component for blood vessel formation. Many signaling receptors expressed in endothelial cells play critical roles in vascular development during embryogenesis. However, downstream response genes required for vascular differentiation are still not clearly identified. Here we describe the development of a protocol for gene-trap expression screening in embryonic stem (ES) cells for endothelial-specific genes. ES cells were differentiated into endothelial cells on an OP9 feeder cell layer in 96-well plates. In a pilot screen, 5 gene-trapped ES cell lines showed an up-regulated expression of the gene trap lacZ reporter out of 864 ES clones screened. One of the trapped genes was endoglin, an endothelial-specific transforming growth factor-beta type III receptor, and another was ASPP1, a p53-binding protein. In vivo expression analysis of the lacZ reporter confirmed that both genes are specifically expressed in endothelial cells during early mouse embryogenesis. Gene-trap expression screening can thus be used to identify early endothelial-specific genes and analyze their function in mice.

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“…The SA-viral vectors are a conditional system used by the German Gene Trap Consortium (http://www.tikus.gsf.de) [31]. Poly-A cell lines are from the Centre for Modeling Human Disease gene-trap project (http://www.cmhd.ca/genetrap) [13]. Poly-A vectors designed to take advantage of nonsense-mediated decay [18] were not included in this analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The stability of the transcript for the antibiotic-resistance gene depends on the poly-A signal from the trapped gene [13]. Because the transcription of the antibiotic-resistance gene product does not depend on the endogenous expression of the trapped gene, poly-A trap vectors are predicted to trap genes regardless of whether the gene is expressed in ESCs.…”

Section: Introductionmentioning

confidence: 99%

Modeling Insertional Mutagenesis Using Gene Length and Expression in Murine Embryonic Stem Cells

et al. 2007

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BackgroundHigh-throughput mutagenesis of the mammalian genome is a powerful means to facilitate analysis of gene function. Gene trapping in embryonic stem cells (ESCs) is the most widely used form of insertional mutagenesis in mammals. However, the rules governing its efficiency are not fully understood, and the effects of vector design on the likelihood of gene-trapping events have not been tested on a genome-wide scale.Methodology/Principal FindingsIn this study, we used public gene-trap data to model gene-trap likelihood. Using the association of gene length and gene expression with gene-trap likelihood, we constructed spline-based regression models that characterize which genes are susceptible and which genes are resistant to gene-trapping techniques. We report results for three classes of gene-trap vectors, showing that both length and expression are significant determinants of trap likelihood for all vectors. Using our models, we also quantitatively identified hotspots of gene-trap activity, which represent loci where the high likelihood of vector insertion is controlled by factors other than length and expression. These formalized statistical models describe a high proportion of the variance in the likelihood of a gene being trapped by expression-dependent vectors and a lower, but still significant, proportion of the variance for vectors that are predicted to be independent of endogenous gene expression.Conclusions/SignificanceThe findings of significant expression and length effects reported here further the understanding of the determinants of vector insertion. Results from this analysis can be applied to help identify other important determinants of this important biological phenomenon and could assist planning of large-scale mutagenesis efforts.

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The Centre for Modeling Human Disease Gene Trap resource

Cited by 28 publications

References 16 publications

Development of a gene‐trap vector with a highly sensitive fluorescent protein reporter system for expression profiling

Development of a gene‐trap vector with a highly sensitive fluorescent protein reporter system for expression profiling

Gene-trap expression screening to identify endothelial-specific genes

Modeling Insertional Mutagenesis Using Gene Length and Expression in Murine Embryonic Stem Cells

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