Controlled Cre/loxP site-specific recombination in the developing brain in medaka fish, Oryzias latipes.
Controlled Cre/loxP site-specific recombination in the developing brain in medaka fish, Oryzias latipes.
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DOI：
10.1371/journal.pone.0066597
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Takeuchi H
中科院分区：
文献类型：
--
作者：
Okuyama T;Isoe Y;Hoki M;Suehiro Y;Yamagishi G;Naruse K;Kinoshita M;Kamei Y;Shimizu A;Kubo T;Takeuchi H
遗传镶嵌技术已被用于可视化和/或遗传修饰各种动物中复杂神经回路内的神经元亚群。然而，可用于马赛克分析的神经群体在脊椎动物大脑中是有限的。为了建立遗传操纵青鳉神经回路的方法，我们首先创建了两个转基因（Tg）青鳉品系，Tg（HSP：Cre）和Tg（HuC：loxP-DsRed-loxP-GFP）。我们证实青鳉HuC启动子衍生的报告基因在青少年青鳉全脑中的表达，并在成年大脑中的神经元前体细胞。然后，我们证明，可以诱导随机重组Cre mRNA显微注射到Tg（HuC：loxP-DsRed-loxP-GFP）胚胎在1-细胞阶段，这使我们能够可视化一些亚群的GFP阳性细胞在成年青鳉脑的端脑区隔化区域。这一发现表明，来自单个或几个祖细胞的克隆相关细胞的分布局限于一个区室化区域。在热循环仪（39°C）中热处理Tg（HSP：Cre X HuC：loxP-DsRed-loxP-GFP）胚胎（受精后0-1天[dpf]）导致全脑中的Cre/loxP重组。与0- 1dpf胚胎相比，2- 3dpf胚胎的重组效率显著降低，表明热诱导重组的敏感性可能依赖于阶段。最后，使用红外激光诱发的基因操作（IR-LEGO）系统，热休克诱导在发育中的大脑中的一个微区域导致可视化的克隆相关的细胞在少年和成年青鳉鱼。我们建立了一个非侵入性的方法来控制Cre/loxP位点特异性重组在青鳉鱼的神经系统发育。这种方法将扩大神经人口马赛克分析，并允许谱系追踪脊椎动物神经系统在青少年和成年阶段。
Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain. To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0–1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2–3 dpf embyos compared with 0–1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish. We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.
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影响因子：
2.5
作者：
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通讯作者：
Kamei, Yasuhiro
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通讯作者：
Yuba, Shunsuke
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2.5
作者：
Kim, CH;Ueshima, E;Miki, N
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