转化(遗传学)
同源重组
生物
基因靶向
基因
遗传学
拟南芥
转基因
拟南芥
核酸内切酶
计算生物学
转基因作物
分子生物学
突变体
作者
Friedrich Fauser,Nadine Roth,Michael Pacher,Gabriele Ilg,Rocı́o Sánchez-Fernández,Christian Biesgen,Holger Puchta
标识
DOI:10.1073/pnas.1202191109
摘要
The development of designed site-specific endonucleases boosted the establishment of gene targeting (GT) techniques in a row of different species. However, the methods described in plants require a highly efficient transformation and regeneration procedure and, therefore, can be applied to very few species. Here, we describe a highly efficient GT system that is suitable for all transformable plants regardless of transformation efficiency. Efficient in planta GT was achieved in Arabidopsis thaliana by expression of a site-specific endonuclease that not only cuts within the target but also the chromosomal transgenic donor, leading to an excised targeting vector. Progeny clonal for the targeted allele could be obtained directly by harvesting seeds. Targeted events could be identified up to approximately once per 100 seeds depending on the target donor combination. Molecular analysis demonstrated that, in almost all events, homologous recombination occurred at both ends of the break. No ectopic integration of the GT vector was found.
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