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Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei

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Abstract

Cre-responsive dual-fluorescent alleles allow in situ marking of cell lineages or genetically modified cells. Here we report a dual-fluorescent allele, ROSA nT-nG, which directs nuclear accumulation of tdTomato in Cre-naïve lineages. Cre converts the allele to ROSA nG, which drives nuclear EGFP accumulation. Conditions were established for analyzing marked nuclei by flow cytometry on the basis of red–green fluorescence and ploidy, with a particular focus on liver nuclei. Hydrodynamic delivery of a Cre-expression plasmid was used to time-stamp arbitrary hepatocytes for lineage tracing. The distinct green fluorescence of nuclei from Cre-exposed lineages facilitated analyses of ploidy transitions within clones. To assess developmental transitions in liver nuclei, ROSA nT-nG was combined with the hepatocyte-specific AlbCre transgene, facilitating discrimination between hepatocyte and nonhepatocyte nuclei. Nuclei extracted from postnatal day 2 (P2) livers were 41 % green and 59 % red and reached a stable level of 84 % green by P22. Until P20, green nuclei were >98 % diploid (2N); at P40 they were ~56 % 2N, 43 % 4N, and <1 % 8N; and by P70 they reached a stable distribution of ~46 % 2N, 45 % 4N, and 9 % 8N. In conclusion, ROSA nT-nG will facilitate in vivo and ex vivo studies on liver and will likely be valuable for studies on tissues like muscle, kidney, or brain in which cells are refractory to whole-cell flow cytometry, or like trophectoderm derivatives or cancers in which cells undergo ploidy transitions.

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Acknowledgments

The authors thank L. Jackiw, J. Hafferman, S. Iverson, K. Thomas, A. Boulet, C. Keller, and the participating Spring-2010 VTMB422 students for their contributions. This work was supported by NIH/NIA grant AG040020 and funds from the Montana Agricultural Experiment Station to EES. Infrastructure support was provided by NIH-COBRE grants GM103394 and GM103500 to Montana State University, and by support from the MSU College of Agriculture and Department of Immunology and Infectious Disease.

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Authors and Affiliations

  1. Molecular Biosciences, IID, Montana State University, Bozeman, MT, 59718, USA

    Justin R. Prigge, James A. Wiley, Emily A. Talago, Elise M. Young, Laura L. Johns, Katherine M. Sonsteng & Edward E. Schmidt

  2. ARC, Montana State University, Tietz Hall, Bozeman, MT, 59718, USA

    Jean A. Kundert

  3. Microbiology and Immunology, Southern Illinois School of Medicine, Springfield, IL, 62794, USA

    William P. Halford

  4. HHMI, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA

    Mario R. Capecchi

  5. Center for Reproductive Biology, Washington State University, Pullman, WA, 99164, USA

    Edward E. Schmidt

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  1. Justin R. Prigge
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Correspondence to Edward E. Schmidt.

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Justin R. Prigge and James A. Wiley have contributed equally to this work.

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335_2013_9469_MOESM3_ESM.tiff

Supplemental Fig. S2. Flow analysis on native mouse liver nuclei. (A & B) Native nuclei were purified from wild-type adult mouse liver, stained with propidium iodide, and analyzed by flow cytometry. In panel A, propidum iodide fluorescence (DNA content) was analyzed by intensity versus peak-width. Intensity showed distinct clustering as a basis of ploidy with roughly 2N increments. Peak-width (X axis) showed that most events in the 6N region of the intensity plot (blue circle) were in wider peaks than events in the 2N, 4N, and 8N region of intensity (boxed and highlighted in red), suggesting that the 6N population were in wider droplets. We interpret this to indicate that the 6N events were predominantly in droplets occupied by more than one nucleus (e.g., a 2N and a 4N, or three 2N, nuclei). The boxed and red-highlighted events in panel A were plotted in panel B to show the distribution of ploidy within this region. 2N, 4N, and 8N events are indicated, showing the relative abundance of the populations. Note that all liver nuclei, including endothelia and other non-hepatocyte cell types are represented here, and all of the non-hepatocyte nuclei will be 2N. (C) Volume of flow cytometry-isolated hepatocyte nuclei as determined by morphometry on slides. DRAQ5- stained nuclei from adult ROSAnT-nG/nT-nG; AlbCre1 mice (hepatocyte nuclei green, other nuclei red) were sorted and the population of green 2N, 4N, and 8N were isolated. Each population was fixed on slides, stained with DAPI, and 150–300 nuclei of each type were analyzed morphometrically. Error bars indicate SEM. (TIFF 824 kb)

335_2013_9469_MOESM4_ESM.tiff

Supplemental Fig. S3. Flow analysis on P175 ROSAnT-nG/+; AlbCre1 mouse liver nuclei. Nuclei were stained with DRAQ5 and analyzed for DRAQ5 fluorescence (DNA content) versus EGFP fluorescence. Hepatocyte nuclei will be green due to expression of AlbCre, whereas non-hepatocyte nuclei will be red. Parameters were as in Fig. 3b of main text. Only two classes of green (i.e., right of black vertical line) diploid nuclei are seen in these cells, and all red (i.e., left of black vertical line) nuclei are diploid (TIFF 706 kb)

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Prigge, J.R., Wiley, J.A., Talago, E.A. et al. Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei. Mamm Genome 24, 389–399 (2013). https://doi.org/10.1007/s00335-013-9469-8

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