Improved Tol2-mediated enhancer trap identifies weakly expressed genes during liver and β cell development and regeneration in zebrafish
Improved Tol2-mediated enhancer trap identifies weakly expressed genes during liver and β cell development and regeneration in zebrafish
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改进的 Tol2 介导的增强子陷阱可识别斑马鱼肝脏和 β 细胞发育和再生过程中的弱表达基因
DOI：
10.1074/jbc.ra118.005568
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发表时间：
2019-01-18
影响因子：
4.8
通讯作者：
Luo, Lingfei
中科院分区：
文献类型：
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作者：
Zhong, Yadong;Huang, Wei;Luo, Lingfei
肝脏和胰腺是两个主要的消化器官，在其中的不同细胞类型中，肝细胞和产生胰岛素的β细胞在健康和疾病中发挥作用。因此，临床医生和研究人员对肝脏和胰腺β细胞的发育和再生机制非常感兴趣。基因和增强子陷阱，如Tol 2转座子为基础的系统是有用的，用于识别基因可能参与斑马鱼模型中的发育过程。在这里，我们开发了一种策略，结合了Tol 2介导的增强子陷阱和Cre/loxP系统，通过使用loxP侧翼的报告驱动的β细胞或肝细胞特异性启动子和上游激活序列（UAS）驱动的Cre。建立了两个双转基因报告细胞系Tg（ins：loxP-CFPNTR-loxP-DsRed; 10 xUAS：Cre，cryaa：Venus）和Tg（fabp 10：loxP-CFPNTR-loxP-DsRed; 10 xUAS：Cre，cryaa：Venus），分别用于标记胰腺β细胞和肝细胞。将这两个双转基因系各自与Tol 2-增强子陷阱建立系杂交，以筛选和鉴定发育期间在β细胞和肝细胞中表达的基因。这种陷阱系统加上应用硝基还原酶（NTR）/甲硝唑（Mtz）介导的细胞消融可以识别再生过程中表达的基因。值得注意的是，先导增强子陷阱捕获瞬时和弱表达的基因，如rab 3da和ensab，其效率高于传统的增强子陷阱系统。总之，通过永久性的基因标记的Cre/loxP，这种改进的Tol 2介导的增强子陷阱系统提供了一个有前途的方法来识别瞬时或弱表达，但潜在的重要，基因在发展和再生。
The liver and pancreas are two major digestive organs, and among the different cell types in them, hepatocytes and the insulin-producing beta cells have roles in both health and diseases. Accordingly, clinicians and researchers are very interested in the mechanisms underlying the development and regeneration of liver and pancreatic beta cells. Gene and enhancer traps such as the Tol2 transposon-based system are useful for identifying genes potentially involved in developmental processes in the zebrafish model. Here, we developed a strategy that combines a Tol2-mediated enhancer trap and the Cre/loxP system by using loxP-flanked reporters driven by beta cell- or hepatocyte-specific promoters and the upstream activating sequence (UAS)-driving Cre. Two double-transgenic reporter lines, Tg(ins:loxP-CFPNTR-loxP-DsRed; 10xUAS:Cre, cryaa:Venus) and Tg(fabp10:loxP-CFPNTR-loxP-DsRed; 10xUAS:Cre, cryaa:Venus), were established to label pancreatic beta cells and hepatocytes, respectively. These two double-transgenic lines were each crossed with the Tol2-enhancer trap founder lines to screen for and identify genes expressed in the beta cell and hepatocytes during development. This trap system coupled with application of nitroreductase (NTR)/metronidazole (Mtz)-mediated cell ablation could identify genes expressed during regeneration. Of note, pilot enhancer traps captured transiently and weakly expressed genes such as rab3da and ensab with higher efficiencies than traditional enhancer trap systems. In conclusion, through permanent genetic labeling by Cre/loxP, this improved Tol2-mediated enhancer trap system provides a promising method to identify transiently or weakly expressed, but potentially important, genes during development and regeneration.