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

Abstract Background The 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. Results Although 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. Conclusions Mosaic 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

Lessons from mouse chimaera experiments with a reiterated transgene marker:revised marker criteria and a review of chimaera markers

Recent reports of a new generation of ubiquitous transgenic chimaera markers prompted us to consider the criteria used to evaluate new chimaera markers and develop more objective assessment methods. To investigate this experimentally we used several series of fetal and adult chimaeras, carrying an older, multi-copy transgenic marker. We used two additional independent markers and objective, quantitative criteria for cell selection and cell mixing to investigate quantitative and spatial aspects of developmental neutrality. We also suggest how the quantitative analysis we used could be simplified for future use with other markers. As a result, we recommend a five-step procedure for investigators to evaluate new chimaera markers based partly on criteria proposed previously but with a greater emphasis on examining the developmental neutrality of prospective new markers. These five steps comprise (1) review of published information, (2) evaluation of marker detection, (3) genetic crosses to check for effects on viability and growth, (4) comparisons of chimaeras with and without the marker and (5) analysis of chimaeras with both cell populations labelled. Finally, we review a number of different chimaera markers and evaluate them using the extended set of criteria. These comparisons indicate that, although the new generation of ubiquitous fluorescent markers are the best of those currently available and fulfil most of the criteria required of a chimaera marker, further work is required to determine whether they are developmentally neutral. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11248-015-9883-7) contains supplementary material, which is available to authorized users

Re-evaluation of the causes of variation among mouse aggregation chimaeras

The composition of adult mouse aggregation chimaeras is much more variable than X-inactivation mosaics. An early theoretical model proposed that almost all the extra variation in chimaeras arises, before X-inactivation occurs, by spatially constrained, geometrical allocation of inner cell mass (ICM) cells to the epiblast and primitive endoderm (PrE). However, this is inconsistent with more recent embryological evidence. Analysis of published results for chimaeric blastocysts and mid-gestation chimaeras suggested that some variation exists among chimaeric morulae and more variation arises both when morula cells are allocated to the ICM versus the trophectoderm (TE) and when ICM cells are allocated to the epiblast versus the PrE. Computer simulation results were also consistent with the conclusion that stochastic allocation of cells to blastocyst lineages in two steps, without the type of geometrical sampling that was originally proposed, could cause a wide variation in chimaeric epiblast composition. Later allocation events will cause additional variation among both chimaeras and X-inactivation mosaics. We also suggest that previously published U-shaped frequency distributions for chimaeric placenta composition might be explained by how TE cells are allocated to the polar TE and/or the subsequent movement of cells from polar TE to mural TE