title: Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei.
abstract: Cre-responsive dual-fluorescent alleles allow in situ marking of cell lineages or genetically modified cells. Here we report a dual-fluorescent allele, ROSAnT-nG, which directs nuclear accumulation of tdTomato in Cre-naïve lineages. Cre converts the allele to ROSAnG, 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, ROSAnT-nGwas 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, ROSAnT-nGwill 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.
authors.affiliation: Molecular Biosciences, IID, Montana State University, Bozeman, MT, 59718, USA.
authors.forename: Justin R
authors.initials: JR
authors.lastname: Prigge
authors.forename: James A
authors.initials: JA
authors.lastname: Wiley
authors.forename: Emily A
authors.initials: EA
authors.lastname: Talago
authors.forename: Elise M
authors.initials: EM
authors.lastname: Young
authors.forename: Laura L
authors.initials: LL
authors.lastname: Johns
authors.forename: Jean A
authors.initials: JA
authors.lastname: Kundert
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Sonsteng
authors.forename: William P
authors.initials: WP
authors.lastname: Halford
authors.forename: Mario R
authors.initials: MR
authors.lastname: Capecchi
authors.forename: Edward E
authors.initials: EE
authors.lastname: Schmidt
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