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Dorer, Douglas R.

doi:10.1023/a:1018460413680

Cited by 53 publications

(6 citation statements)

References 51 publications

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“…The underlying clone has been isolated at Novo Nordisk (Copenhagen) by a routine screening procedure of transfected clones and was used with kind permission. It was chosen because it harbors a single copy of the FVII c-DNA gene (C. Bornaes, private communication), in contrast to many examples where high producers rely on multiple copy integration events which may cause long-term problems due to epigenetic inactivation events or genomic instability (33,34).…”

Section: Resultsmentioning

confidence: 99%

“…Specific targeting of well-characterized genomic sites can solve major problems associated with current gene-transfer techniques. Traditionally, these techniques tend to yield variable expression levels due to chromosomal position effects, and they may be severely affected by unpredictable cross interactions between the multiple copies which are usually integrated (33,34). If the vector is transferred as a circular plasmid, the site of its opening cannot be controlled.…”

Section: Discussionmentioning

confidence: 99%

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Retargeting of Retroviral Integration Sites for the Predictable Expression of Transgenes and the Analysis of Cis-Acting Sequences¹

Schübeler¹,

Maass²,

Bode³

1998

Biochemistry

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The transcriptional activity of transgenes in eukaryotic host cells critically depends on the sites of their integration where it is modulated by interactions between the promoter and surrounding chromatin structures. Retroviruses integrate their genome into chromosomal loci favoring expression from one long-terminal repeat (LTR). We have therefore developed a strategy in which retroviral vectors are provided with "tags", that is, targets for a site-specific recombinase (Flp). Presence of two 48 bp Flp recognition target (FRT) sequences permits the excision of a selection marker whereby the reading frame of a reporter gene (lacZ) is restored and beta-galactosidase activity can be monitored to characterize the integration site regarding the level and stability of expression. The location of the remaining FRT tag permits the subsequent Flp-mediated insertion of a truncated selection marker which is then expressed from the LTR. This step represents an authentic site-specific recombination event which can be demonstrated by a number of criteria, among these its reversibility in the presence of Flp activity. Thereby the "expression trap" principle permits the efficient recovery of stable insertion events, and if a gene of interest is linked to the truncated marker, the established properties of a given genomic site can be utilized for transcription studies or for the generation of highly expressing clones, even for biotechnological purposes.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Retargeting of Retroviral Integration Sites for the Predictable Expression of Transgenes and the Analysis of Cis-Acting Sequences¹

Schübeler¹,

Maass²,

Bode³

1998

Biochemistry

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“…Indirect evidence suggested that mosaic expression of a different transgene in the mouse adrenal cortex involved stochastic silencing at the level of the chromosome or transgene, rather than at the cellular level [44] but this may not apply to all mosaic transgenic lines. Transgene silencing can occur if the transgene integrates near an endogenous heterochromatic site [41], which may prevent the transcriptional machinery from accessing the transgene, or if it triggers the formation of heterochromatin by integrating as a series of repeated copies [45, 46]. Repeat-copy transgenes are also associated with methylation changes that could mediate stochastic transgene silencing if transgene methylation levels vary among cells [47].…”

Section: Discussionmentioning

confidence: 99%

Comparison of two related lines of tauGFP transgenic mice designed for lineage tracing

et al. 2017

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BackgroundThe tauGFP reporter fusion protein is produced nearly ubiquitously by the TgTP6.3 transgene in TP6.3 mice and its localisation to microtubules offers some advantages over soluble GFP as a lineage marker. However, TgTP6.3 Tg/Tg homozygotes are not viable and TgTP6.3 Tg/− hemizygotes are smaller than wild-type. TP6.4 mice carry the TgTP6.4 transgene, which was produced with the same construct used to generate TgTP6.3, so we investigated whether TgTP6.4 had any advantages over TgTP6.3.ResultsAlthough TgTP6.4 Tg/Tg homozygotes died before weaning, TgTP6.4 Tg/− hemizygotes were viable and fertile and only males were significantly lighter than wild-type. The TgTP6.4 transgene produced the tauGFP fusion protein by the 2-cell stage and it was widely expressed in adults but tauGFP fluorescence was weak or absent in several tissues, including some neural tissues. The TgTP6.4 transgene expression pattern changed over several years of breeding and mosaic transgene expression became increasingly common in all expressing tissues. This mosaicism was used to visualise clonal lineages in the adrenal cortex of TgTP6.4 Tg/− hemizygotes and these were qualitatively and quantitatively comparable to lineages reported previously for other mosaic transgenic mice, X-inactivation mosaics and chimaeras. Mosaicism occurred less frequently in TP6.3 than TP6.4 mice and was only observed in the corneal epithelium and adrenal cortex.ConclusionsMosaic expression makes the TgTP6.4 transgene unsuitable for use as a conventional cell lineage marker but such mosaicism provides a useful system for visualising clonal lineages that arise during development or maintenance of adult tissues. Differences in the occurrence of mosaicism between related transgenic lines, such as that described for lines TP6.3 and TP6.4, might provide a useful system for investigating the mechanism of transgene silencing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-017-0149-x) contains supplementary material, which is available to authorized users.

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“…Prokaryotic sequences -especially their CpG dinucleotide base pairs -may undergo methylation or heterochromatin formation in animal cells, which leads to transgene silencing. It is becoming increasingly clear that silenced transgenes have been recognized as foreign elements by host cellular mechanisms, as have retroviral and transposon sequences [85,86]. Characteristics of the transgenes used for different gene-delivery methods and gene-transfer programs in farm animals.…”

Section: Transgenes -Gene Constructsmentioning

confidence: 99%

Transgenic Modification of Production Traits in Farm Animals

Brem¹,

Müller²

2006

Genetically Engineered Food

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Untitled

Cited by 53 publications

References 51 publications

Retargeting of Retroviral Integration Sites for the Predictable Expression of Transgenes and the Analysis of Cis-Acting Sequences¹

Retargeting of Retroviral Integration Sites for the Predictable Expression of Transgenes and the Analysis of Cis-Acting Sequences¹

Comparison of two related lines of tauGFP transgenic mice designed for lineage tracing

Transgenic Modification of Production Traits in Farm Animals

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Untitled

Cited by 53 publications

References 51 publications

Retargeting of Retroviral Integration Sites for the Predictable Expression of Transgenes and the Analysis of Cis-Acting Sequences1

Retargeting of Retroviral Integration Sites for the Predictable Expression of Transgenes and the Analysis of Cis-Acting Sequences1

Comparison of two related lines of tauGFP transgenic mice designed for lineage tracing

Transgenic Modification of Production Traits in Farm Animals

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Retargeting of Retroviral Integration Sites for the Predictable Expression of Transgenes and the Analysis of Cis-Acting Sequences¹

Retargeting of Retroviral Integration Sites for the Predictable Expression of Transgenes and the Analysis of Cis-Acting Sequences¹