β-globin matrix attachment region improves stable genomic expression of the Sleeping Beauty transposon.
β-globin matrix attachment region improves stable genomic expression of the Sleeping Beauty transposon.
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DOI：
10.1002/jcb.23159
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发表时间：
2011-09
影响因子：
4
通讯作者：
Kren, Betsy T.
中科院分区：
文献类型：
--
作者：
Sjeklocha, Lucas;Chen, Yixin;Daly, Meghan C.;Steer, Clifford J.;Kren, Betsy T.
肝脏是一个有吸引力的基因治疗靶点，因为它具有广泛的蛋白质生产能力，并且由于它通常提供的基因功能的丧失而导致许多疾病。睡美人转座子（SB-Tn）1系统是一种非病毒载体，能够传递和介导治疗性转基因插入到宿主基因组中进行长期表达。目前该系统面临的一个挑战是转基因整合效率低。在这项研究中，我们使用人肝癌细胞系（HuH-7）和原代人造血内皮细胞（BOECs）来测试含有DNA元素的载体，以增强转位而不整合自身。我们利用人β-珠蛋白基质附着区（MAR）和猿猴病毒40 （SV40）核转位信号，将每个元件持续表达GFP::Zeo报告结构的HuH-7细胞的百分比提高了约50%；而两者联合使用则没有叠加效应。有趣的是，这两个元素一起对插入位点的数量表现出加性效应，而在boec中，SV40单独似乎对转位有抑制作用。在长期培养中，质粒DNA的丢失，转座酶的表达和插入位点的定位证明了真正的转座而没有附带表达。这些结果表明，在SB-Tn系统的主干中添加β-珠蛋白MAR和潜在的其他元件可以增强治疗性转基因的转位和表达。这些发现可能对利用SB转基因给肝治疗多种疾病产生重大影响。
The liver is an attractive target for gene therapy due to its extensive capability for protein production and the numerous diseases resulting from a loss of gene function it normally provides. The Sleeping Beauty Transposon (SB-Tn)1 system is a non-viral vector capable of delivering and mediating therapeutic transgene(s) insertion into the host genome for long-term expression. A current challenge for this system is the low efficiency of integration of the transgene. In this study we use a human hepatoma cell line (HuH-7) and primary human blood outgrowth endothelial cells (BOECs) to test vectors containing DNA elements to enhance transposition without integrating themselves. We employed the human β-globin matrix attachment region (MAR) and the Simian virus 40 (SV40) nuclear translocation signal to increase the percent of HuH-7 cells persistently expressing a GFP::Zeo reporter construct by ~50% for each element; while combining both did not show an additive effect. Interestingly, both elements together displayed an additive effect on the number of insertion sites, and in BOECs the SV40 alone appeared to have an inhibitory effect on transposition. In long-term cultures the loss of plasmid DNA, transposase expression and mapping of insertion sites demonstrated bona fide transposition without episomal expression. These results show that addition of the β-globin MAR and potentially other elements to the backbone of SB-Tn system can enhance transposition and expression of therapeutic transgenes. These findings may have a significant influence on the use of SB transgene delivery to liver for the treatment of a wide variety of disorders.
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影响因子：
12.4
作者：
Chen, Zhi-Ying;Riu, Efren;Kay, Mark A.
通讯作者：
Kay, Mark A.
DOI：
10.1385/1-59259-755-6:255
发表时间：
2004-01-01
期刊：
MOBILE GENETIC ELEMENTS: PROTOCOLS AND GENOMIC APPLICATIONS
影响因子：
--
作者：
Ivics, Z;Izsvák, Z
通讯作者：
Izsvák, Z
影响因子：
12.4
作者：
Geurts, AM;Yang, Y;Hackett, PB
通讯作者：
Hackett, PB
影响因子：
12.4
作者：
Baus, J;Liu, L;Fletcher, BS
通讯作者：
Fletcher, BS
影响因子：
56.9
作者：
BODE, J;KOHWI, Y;KOHWISHIGEMATSU, T
通讯作者：
KOHWISHIGEMATSU, T