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

Abstract

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.

FxClear, A Free-hydrogel Electrophoretic Tissue Clearing Method for Rapid De-lipidation of Tissues with High Preservation of Immunoreactivity

“…5F and Supplementary Video 1). This is very similar to the tau-GFP expression reported in this developmental stage [31]. Collectively, these data demonstrated that various thick organs can be cleared with the FxClear protocol, despite the concerns about limited tissue transparency and potential loss of proteins.…”

Selection against BALB/c strain cells in mouse chimaeras

“…The tauGFP marker labelled the partner (BF1×TP6.3) embryos rather than the BALB/c embryos in series 4U chimaeras, or the AAF2 embryos in series 4B chimaeras. To allow all the chimaeric blastocysts to be analysed at equivalent stages, they were cultured in an environmental chamber on the stage of a confocal microscope ( Sharp et al, 2017 ) and imaged at intervals ( Fig. 1 ).…”

“…For the experiments with GFP blastocyst chimaeras, embryos were aggregated, cultured in KSOM medium ( Summers et al, 1995 ) under mineral oil in an incubator (37°C, 5% CO 2 in air) for 24 h, then transferred to fresh pre-equilibrated drops of culture medium under mineral oil in a WillCo HBSt-3522, thin glass-bottomed dish (Intracel Ltd., Royston, Herts, UK). This was placed in an environmental chamber on top of the pre-heated stage (THD 60, Linkam Scientific Instruments Ltd., Tadworth, UK) of a Leica DMIRB/E inverted confocal microscope, and the atmosphere within the chamber was maintained at 37°C, 5% CO 2 in air, as described elsewhere ( Sharp et al, 2017 ). Time-lapse images were acquired using the Leica TCS NT confocal system, and images from fluorescein isothiocyanate (FITC) and transmitted light channels were merged ( Sharp et al, 2017 ).…”

“…This was placed in an environmental chamber on top of the pre-heated stage (THD 60, Linkam Scientific Instruments Ltd., Tadworth, UK) of a Leica DMIRB/E inverted confocal microscope, and the atmosphere within the chamber was maintained at 37°C, 5% CO 2 in air, as described elsewhere ( Sharp et al, 2017 ). Time-lapse images were acquired using the Leica TCS NT confocal system, and images from fluorescein isothiocyanate (FITC) and transmitted light channels were merged ( Sharp et al, 2017 ). The percentage contributions of tauGFP-positive cells to the inner cell mass (ICM), polar trophectoderm (pTE; overlying the ICM) and mural trophectoderm (mTE; surrounding the blastocyst cavity) were estimated in chimaeric blastocysts.…”