Conditional, recombinase-mediated expression of genes in plant cell cultures
Conditional, recombinase-mediated expression of genes in plant cell cultures
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DOI：
10.1111/j.1365-313x.2004.02004.x
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发表时间：
2004-03-01
期刊：
影响因子：
7.2
通讯作者：
De Veylder, L
中科院分区：
文献类型：
--
作者：
Joubès, J;De Schutter, K;De Veylder, L
在植物细胞中，关键基因的过度表达可能会受到对发育的有害影响的阻碍，从而导致含有感兴趣基因的转基因细胞的反选择。已经报道了诱导表达系统，但许多系统存在不必要的泄漏表达。为了避免这个潜在的问题，一种新的诱导系统基于两个先前表征的系统：噬菌体P1的CRE-loxP位点特异性重组系统和哺乳动物糖皮质激素受体（GR）的亚细胞靶向蛋白。通过将大鼠GR的受体结构域融合到CRE重组酶的羧基端，建立了一个双锁条件转录诱导系统，该系统对表达可能阻断转基因再生的基因过表达非常有用。此外，所设计的载体采用GATEWAY（TM）重组技术，克隆无限制性和结扎性，适合高通量研究。该系统在烟草亮黄2 （BY-2）细胞中进行了测试，其效率被证明是gus报告基因和a型细胞周期蛋白依赖性激酶（CDKA）突变等位基因的控制过表达，CDKA是一种有效的细胞周期抑制剂。
In plant cells, overexpression of critical genes can be hampered by deleterious effects on development that results in a counterselection of transgenic cells harboring the gene of interest. Inducible expression systems have been reported, but many of them show unwanted leaky expression. To circumvent this potential problem, a novel inducible system was developed based on two previously characterized systems: the CRE-loxP site-specific recombination system of bacteriophage P1 and the subcellular targeting of proteins by a mammalian glucocorticoid receptor (GR). By fusing the receptor domain of the rat GR to the carboxyl terminus of the CRE recombinase, a double-lock conditional transcriptional induction system was created that is highly useful to overexpress genes whose expression may block transgenic regeneration. Furthermore, because the designed vector utilizes the GATEWAY(TM) recombination technology, cloning was restriction- and ligation-free, thus rendering the vector suitable for high-throughput research. The system was tested in Nicotiana tabacum bright yellow-2 (BY-2) cells and its efficiency was demonstrated for the controlled overexpression of the gus reporter gene and a mutant allele of the A-type cyclin-dependent kinase (CDKA), which is known to be a potent inhibitor of the cell cycle.