High-level genomic integration, epigenetic changes, and expression of sleeping beauty transgene.
High-level genomic integration, epigenetic changes, and expression of sleeping beauty transgene.
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高水平基因组整合、表观遗传变化和睡美人转基因表达。
DOI：
10.1021/bi9016846
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发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Steer,CliffordJ
中科院分区：
文献类型：
--
作者：
Zhu,Jianhui;Park,ChangWon;Sjeklocha,Lucas;Kren,BetsyT;Steer,CliffordJ
睡美人转座子（Sleeping Beautytransposon，SB-Tn）是一种重要的非病毒载体，可将转基因整合到哺乳动物基因组中。我们在这里报告了一种新的双荧光标记SB-Tn系统，该系统允许非选择性荧光激活细胞分选SB-Tn转导的K562红系细胞。利用内部核糖体进入位点元件，将绿色荧光蛋白（eGFP）连接到SB 10转座酶基因上，作为SB 10转座酶稳定表达的间接标记。通过eGFP进行的荧光激活细胞分选（FACS）导致显著富集（>60%）的细胞，其具有SB-Tn介导的基因组插入和DsRed转基因的长期表达。DsRed转基因的杂合红系特异性启动子在K562细胞的红系或巨核细胞分化中得到验证。亚硫酸氢盐介导的基因组分析确定了DsRed+和DsRed−细胞克隆之间不同的DNA甲基化模式，表明在转基因表达中起着关键作用。此外，尽管启动子元件的宿主基因组拷贝未显示CpG甲基化，但转基因所携带的相同序列明显高甲基化。另外的证据也表明组蛋白去乙酰化在DsRed转基因的调节中的作用。SB转基因的存在影响了距离>45 kb的邻近宿主基因的表达。我们的数据表明，一个荧光标记SB-Tn系统可用于富集哺乳动物细胞harboringSB介导的转基因插入。观察到的表观遗传变化也表明，SB插入的转基因可以被内源性因素选择性地修饰。此外，宿主基因的长距离激活现在必须被认为是插入的含有增强子元件的转基因盒的潜在后果。
Sleeping Beautytransposon (SB-Tn) has emerged as an important nonviral vector for integrating transgenes into mammalian genomes. We report here a novel dual fluorescent reportercis SB-Tn system that permitted nonselective fluorescent-activated cell sorting forSB-Tn-transduced K562 erythroid cells. Using an internal ribosome entry site element, the green fluorescent protein (eGFP) was linked to theSB10 transposase gene as an indirect marker for the robust expression ofSB10 transposase. Flourescence-activated cell sorting (FACS) by eGFP resulted in significant enrichment (>60%) of cells exhibitingSB-Tn-mediated genomic insertions and long-term expression of a DsRed transgene. The hybrid erythroid-specific promoter of DsRed transgene was verified in erythroid or megakaryocyte differentiation of K562 cells. Bisulfite-mediated genomic analyses identified different DNA methylation patterns between DsRed+and DsRed−cell clones, suggesting a critical role in transgene expression. Moreover, although the host genomic copy of the promoter element showed no CpG methylation, the same sequence carried by the transgene was markedly hypermethylated. Additional evidence also suggested a role for histone deacetylation in the regulation of DsRed transgene. The presence ofSBtransgene affected the expression of neighboring host genes at distances >45 kb. Our data suggested that a fluorescent reportercis SB-Tn system can be used to enrich mammalian cells harboringSB-mediated transgene insertions. The observed epigenetic changes also demonstrated that transgenes inserted bySBcould be selectively modified by endogenous factors. In addition, long-range activation of host genes must now be recognized as a potential consequence of an inserted transgene cassette containing enhancer elements.