Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei
Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei
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DOI：
10.1007/s00335-013-9469-8
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发表时间：
2013-10-01
期刊：
影响因子：
2.5
通讯作者：
Schmidt, Edward E.
中科院分区：
文献类型：
--
作者：
Prigge, Justin R.;Wiley, James A.;Schmidt, Edward E.
Cre反应的双荧光等位基因允许对细胞系或转基因细胞进行原位标记。在这里，我们报告了一个双荧光等位基因ROSA(NT-Ng)，它指示td番茄在Cre-na和Lt；ve系谱系中的核积累。Cre将等位基因转化为ROSA(Ng)，从而驱动核内EGFP的积累。建立了基于红绿色荧光和倍体的流式细胞术分析标记核的条件，特别是肝细胞核。Cre表达载体的流体动力传递被用来对任意肝细胞进行时间标记以进行谱系追踪。来自CRE暴露谱系的核的明显绿色荧光有助于分析克隆内的倍性转变。为了评估肝细胞核的发育转变，ROSA(NT-Ng)与肝细胞特异性的AlbCre转基因基因相结合，促进了肝细胞核和非肝细胞核的区分。从出生后第2天(P2)的肝脏提取的细胞核有41%是绿色的，59%是红色的，到22d时达到稳定的水平，84%是绿色的。至P20，绿核为98%的二倍体(2N)，P40与56%2N、43%4N和1%8N相似，至P70达到稳定分布，分别为46%2N、45%4N和9%8N。总之，ROSA(NT-Ng)将促进肝脏的体内和体外研究，并可能对肌肉、肾脏或脑等组织的研究有价值，这些组织中的细胞对全细胞流式细胞术是不敏感的，或者像滋养外胚层的衍生物或细胞经历倍体转变的癌症。
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-na < ve 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 similar to 56 % 2N, 43 % 4N, and < 1 % 8N; and by P70 they reached a stable distribution of similar to 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.