Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei
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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.
KeywordsLiver Nucleus Hepatocyte Nucleus ROSA26 Locus Fluorescent Nucleus Green Nucleus
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.
- Fung YC (2000) Biomechanics: motion, flow, stress, and growth. Springer, New YorkGoogle Scholar
- Postic C, Shiota M, Niswender KD, Jetton TL, Chen Y, Moates JM, Shelton KD, Lindner J, Cherrington AD, Magnuson MA (1999) Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 274:305–315PubMedCrossRefGoogle Scholar