Differentiation trapping screen in live culture for genes expressed in cardiovascular lineages

Chen, Weisheng V.; Chen, Zhi

doi:10.1002/dvdy.10427

Cited by 6 publications

(3 citation statements)

References 46 publications

(42 reference statements)

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“…axon tracts) is the placental alkaline phosphatase (PLAP; (Leighton et al, 2001)). For live imaging of gene expression, fluorescent proteins previously used in gene trap vectors include green fluorescent protein (GFP) (Ishida and Leder, 1999), Venus (Tanaka et al, 2008), and fusions of GFP to neo (Chen and Chen, 2004) or β-geo (Tsakiridis et al, 2007). …”

Section: Design Of the Gene Trap Vectormentioning

confidence: 99%

Gene Trap Mutagenesis in the Mouse

Friedel

Soriano

2010

Methods in Enzymology

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Gene trapping in mouse embryonic stem (ES) cells is an efficient method for the mutagenesis of the mammalian genome. Insertion of a gene trap vector disrupts gene function, reports gene expression, and provides a convenient tag for the identification of the insertion site. The trap vector can be delivered to ES cells by electroporation of a plasmid, by retroviral infection, or by transposon-mediated insertion. Recent developments in trapping technology involve the utilization of site-specific recombination sites, which allow the induced modification of trap alleles in vitro and in vivo. Gene trapping strategies have also been successfully developed to screen for genes that are acting in specific biological pathways. In this chapter, we review different applications of gene trapping, and we provide detailed experimental protocols for gene trapping in ES cells by retroviral and transposon gene trap vectors.

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Section: Design Of the Gene Trap Vectormentioning

confidence: 99%

Gene Trap Mutagenesis in the Mouse

Friedel

Soriano

2010

Methods in Enzymology

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“…The ease of using the gene trap approach and GFP FACS profiling, plus the simplicity of identifying the trapped genes, make this vector a suitable choice to target and isolate ES cell-specific genes in mice. Similar to previous studies [ 17 - 22 ], it can be applied to other cell types to identify developmentally regulated genes or genes that are responsive to certain growth factors.…”

Section: Introductionmentioning

confidence: 98%

Identifying genes preferentially expressed in undifferentiated embryonic stem cells

Leder

2007

BMC Cell Biol

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BackgroundThe mechanism involved in the maintenance and differentiation of embryonic stem (ES) cells is incompletely understood.ResultsTo address this issue, we have developed a retroviral gene trap vector that can target genes expressed in undifferentiated ES cells. This gene trap vector harbors both GFP and Neo reporter genes. G-418 drug resistance was used to select ES clones in which the vector was integrated into transcriptionally active loci. This was then followed by GFP FACS profiling to identify ES clones with reduced GFP fluorescence and, hence, reduced transcriptional activity when ES cells differentiate. Reduced expression of the GFP reporter in six of three hundred ES clones in our pilot screening was confirmed to be down-regulated by Northern blot analysis during ES cell differentiation. These six ES clones represent four different genes. Among the six integration sites, one was at Zfp-57 whose gene product is known to be enriched in undifferentiated ES cells. Three were located in an intron of a novel isoform of CSL/RBP-Jkappa which encodes the key transcription factor of the LIN-12/Notch pathway. Another was inside a gene that may encode noncoding RNA transcripts. The last integration event occurred at a locus that may harbor a novel gene.ConclusionTaken together, we demonstrate the use of a novel retroviral gene trap vector in identifying genes preferentially expressed in undifferentiated ES cells.

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“…On the other hand, fluorescent proteins such as the enhanced green fluorescent protein (EGFP) are now widely utilized as reporters to study gene expression in real time. To date, only a few groups have used fluorescent reporters to monitor expression of trapped genes (Chen and Chen, 2004;Ishida and Leder, 1999;Medico et al, 2001;Tsakiridis A. et al, 2007). This is primarily because detection of gene expression by EGFP is less sensitive than by b-gal staining.…”

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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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Differentiation trapping screen in live culture for genes expressed in cardiovascular lineages

Cited by 6 publications

References 46 publications

Gene Trap Mutagenesis in the Mouse

Gene Trap Mutagenesis in the Mouse

Identifying genes preferentially expressed in undifferentiated embryonic stem cells

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

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