Zinc finger nuclease-mediated transgene deletion
Zinc finger nuclease-mediated transgene deletion
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DOI：
10.1007/s11103-010-9641-4
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发表时间：
2010-08-01
影响因子：
5.1
通讯作者：
Russell, Sean
中科院分区：
文献类型：
--
作者：
Petolino, Joseph F.;Worden, Andrew;Russell, Sean
通过将稳定转化的植物与表达相应 ZFN 基因的第二种植物杂交，从稳定转化的植物中删除侧翼为锌指核酸酶 (ZFN) 切割位点的转基因。将含有侧翼为 ZFN 切割位点的 GUS 报告基因、GFP 报告基因和 PAT 选择标记基因的目标构建体转化到烟草中。再生Basta(A(R))抗性植物并筛选GUS和GFP表达。含有由组成型 CsVMV 启动子驱动的 ZFN 基因和 HPT 选择标记基因的第二个构建体也被转化到烟草中。选定的T-0植物生长至成熟并允许自花授粉。表达GUS和GFP的纯合目标植物与表达ZFN基因的纯合ZFN植物杂交。在杂种中观察到许多 GUS 阴性植物，其中一种特定的杂交显示与 35% GUS 阴性植物相似。获得了包含 GUS 基因的 4.3 kb 序列完全缺失的证据并确认了序列。在 F-2 后代中观察到“截短的”和“完整的”靶序列与预期报告基因表型的共分离。由于 ZFN 可以设计用于结合和切割多种 DNA 序列，因此这些结果构成了创建目标基因删除的通用策略。
A transgene, flanked by zinc finger nuclease (ZFN) cleavage sites, was deleted from a stably transformed plant by crossing it with a second plant expressing a corresponding ZFN gene. A target construct, containing a GUS reporter gene flanked by ZFN cleavage sites, a GFP reporter gene and a PAT selectable marker gene, was transformed into tobacco. Basta(A (R))-resistant plants were regenerated and screened for GUS and GFP expression. A second construct, containing a ZFN gene driven by the constitutive CsVMV promoter and an HPT selectable marker gene, was also transformed into tobacco. Selected T-0 plants were grown to maturity and allowed to self-pollinate. Homozygous target plants, which expressed GUS and GFP, were crossed with homozygous ZFN plants, which expressed the ZFN gene. Numerous GUS-negative plants were observed among the hybrids with one particular cross displaying similar to 35% GUS-negative plants. Evidence for complete deletion of a 4.3 kb sequence comprising the GUS gene was obtained and sequence confirmed. Co-segregation in F-2 progenies of 'truncated' and 'intact' target sequences with expected reporter gene phenotypes were observed. Since ZFNs can be designed to bind and cleave a wide range of DNA sequences, these results constitute a general strategy for creating targeted gene deletions.