Liu Qizhi, Wang Yunzhi, Lin Fan, Zhang Lei, Li Yan, Ge Ruowen, Hong Yunhan
Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
PLoS One. 2015 Jun 1;10(6):e0127961. doi: 10.1371/journal.pone.0127961. eCollection 2015.
Retrovirus (RV) is efficient for gene transfer and integration in dividing cells of diverse organisms. RV provides a powerful tool for insertional mutagenesis (IM) to identify and functionally analyze genes essential for normal and pathological processes. Here we report RV-mediated gene transfer and genome-wide IM in fish stem cells from medaka and zebrafish. Three RVs were produced for fish cell transduction: rvLegfp and rvLcherry produce green fluorescent protein (GFP) and mCherry fluorescent protein respectively under control of human cytomegalovirus immediate early promoter upon any chromosomal integration, whereas rvGTgfp contains a splicing acceptor and expresses GFP only upon gene trapping (GT) via intronic in-frame integration and spliced to endogenous active genes. We show that rvLegfp and rvLcherry produce a transduction efficiency of 1123% in medaka and zebrafish stem cell lines, which is as 3067% efficient as the positive control in NIH/3T3. Upon co-infection with rvGTgfp and rvLcherry, GFP-positive cells were much fewer than Cherry-positive cells, consistent with rareness of productive gene trapping events versus random integration. Importantly, rvGTgfp infection in the medaka haploid embryonic stem (ES) cell line HX1 generated GTgfp insertion on all 24 chromosomes of the haploid genome. Similar to the mammalian haploid cells, these insertion events were presented predominantly in intergenic regions and introns but rarely in exons. RV-transduced HX1 retained the ES cell properties such as stable growth, embryoid body formation and pluripotency gene expression. Therefore, RV is proficient for gene transfer and IM in fish stem cells. Our results open new avenue for genome-wide IM in medaka haploid ES cells in culture.
逆转录病毒（RV）在多种生物的分裂细胞中进行基因转移和整合的效率很高。RV为插入诱变（IM）提供了一个强大的工具，用于识别和功能分析正常和病理过程中必需的基因。在此，我们报告了RV介导的青鳉和斑马鱼鱼类干细胞中的基因转移和全基因组IM。制备了三种用于鱼类细胞转导的RV：rvLegfp和rvLcherry在任何染色体整合后，在人巨细胞病毒立即早期启动子的控制下分别产生绿色荧光蛋白（GFP）和mCherry荧光蛋白，而rvGTgfp含有一个剪接受体，仅在通过内含子框内整合进行基因捕获（GT）并剪接到内源性活性基因时才表达GFP。我们表明，rvLegfp和rvLcherry在青鳉和斑马鱼干细胞系中的转导效率为11%至23%，与NIH/3T3中的阳性对照效率相当，为30%至67%。与rvGTgfp和rvLcherry共感染后，GFP阳性细胞比Cherry阳性细胞少得多，这与生产性基因捕获事件相对于随机整合的稀有性一致。重要的是，rvGTgfp在青鳉单倍体胚胎干细胞系HX1中的感染在单倍体基因组的所有24条染色体上产生了GTgfp插入。与哺乳动物单倍体细胞类似，这些插入事件主要出现在基因间区域和内含子中，但很少出现在外显子中。RV转导的HX1保留了ES细胞的特性，如稳定生长、胚状体形成和多能性基因表达。因此，RV在鱼类干细胞中擅长基因转移和IM。我们的结果为培养中的青鳉单倍体ES细胞的全基因组IM开辟了新途径。
