Kato Hiroyuki, Abe Kota, Yokota Shinpei, Matsuno Rinta, Mikekado Tsuyoshi, Yokoi Hayato, Suzuki Tohru
Laboratory of Marine Life Science and Genetics, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan,
In Vitro Cell Dev Biol Anim. 2015 Jan;51(1):42-9. doi: 10.1007/s11626-014-9805-7. Epub 2014 Dec 17.
The establishment of induced pluripotent stem (iPS) cell technology in fish could facilitate the establishment of novel cryopreservation techniques for storing selected aquaculture strains as frozen cells. In order to apply iPS cell technology to fish, we established a transgenic zebrafish line, Tg(Tru.oct4:EGFP), using green fluorescent protein (GFP) expression under the control of the oct4 gene promoter as a marker to evaluate multipotency in iPS cell preparations. We used the oct4 promoter from fugu (Takifugu rubripes) due to the compact nature of the fugu genome and to facilitate future applications of this technology in marine fishes. During embryogenesis, maternal GFP fluorescence was observed at the cleavage stage and zygotic GFP expression was observed from the start of the shield stage until approximately 24 h after fertilization. gfp messenger RNA (mRNA) was expressed by whole embryonic cells at the shield stage, and then restricted to the caudal neural tube in the latter stages of embryogenesis. These observations showed that GFP fluorescence and the regulation of gfp mRNA expression by the exogenous fugu oct4 promoter are well suited for monitoring endogenous oct4 mRNA expression in embryos. Bisulfite sequencing revealed that the rate of CpG methylation in the transgenic oct4 promoter was high in adult cells (98%) and low in embryonic cells (37%). These findings suggest that, as with the endogenous oct4 promoter, demethylation and methylation both take place normally in the transgenic oct4 promoter during embryogenesis. The embryonic cells harvested at the shield stage formed embryonic body-like cellular aggregates and maintained GFP fluorescence for 6 d when cultured on Transwell-COL Permeable Supports or a feeder layer of adult fin cells. Loss of GFP fluorescence by cultured cells was correlated with cellular differentiation. We consider that the Tg(Tru.oct4:EGFP) zebrafish line established here is well suited for monitoring multipotency in multipotent zebrafish cell cultures and for iPS cell preparation.
鱼类中诱导多能干细胞（iPS）技术的建立有助于开发新的冷冻保存技术，将选定的水产养殖品系作为冷冻细胞保存。为了将iPS细胞技术应用于鱼类，我们建立了一个转基因斑马鱼品系Tg(Tru.oct4:EGFP)，利用在oct4基因启动子控制下的绿色荧光蛋白（GFP）表达作为标记，来评估iPS细胞制备中的多能性。我们使用了来自河豚（红鳍东方鲀）的oct4启动子，这是由于河豚基因组结构紧凑，便于该技术未来在海水鱼类中的应用。在胚胎发育过程中，在卵裂期观察到母源GFP荧光，从盾形期开始直至受精后约24小时观察到合子GFP表达。在盾形期，整个胚胎细胞都表达gfp信使核糖核酸（mRNA），然后在胚胎发育后期局限于尾神经管。这些观察结果表明，GFP荧光以及外源河豚oct4启动子对gfp mRNA表达的调控非常适合监测胚胎中内源性oct4 mRNA的表达。亚硫酸氢盐测序显示，转基因oct4启动子中CpG甲基化率在成体细胞中较高（98%），而在胚胎细胞中较低（37%）。这些发现表明，与内源性oct4启动子一样，在胚胎发育过程中，转基因oct4启动子的去甲基化和甲基化过程均正常发生。在盾形期收获的胚胎细胞在Transwell-COL可渗透支持物或成年鳍细胞饲养层上培养时，形成了胚胎体样细胞聚集体，并保持GFP荧光长达6天。培养细胞中GFP荧光的丧失与细胞分化相关。我们认为，这里建立的Tg(Tru.oct4:EGFP)斑马鱼品系非常适合监测多能斑马鱼细胞培养中的多能性以及用于iPS细胞制备。
