Site‐specific gene integration in rice genome mediated by the FLP–FRT recombination system

Abstract

SummaryPlant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombination systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT system for site-specific gene integration in plants using rice as a model.

Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants

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QTL analysis reveals the genetic architecture of domestication traits in Crisphead lettuce

“…A transgene placed in close linkage with a gene or genomic block that causes a lower fitness in the wild habitat is likely to be purged from the wild populations (Gressel 1999;Stewart et al 2003). Techniques for targeted insertion of transgenes to specific regions in the genome are currently being developed (Lombardo et al 2011;Nandy and Srivastava 2011;Shukla et al 2009). Based on these greenhouse results, the clustering regions on LG3 and LG7 may be considered as such possible insertion sites.…”

Generation of marker‐free transgenic hexaploid wheat via an Agrobacterium‐mediated co‐transformation strategy in commercial Chinese wheat varieties

“…It is considered important to generate marker‐free varieties where the marker gene used for the generation of positive transgenic plants has been eliminated. Marker‐free transgenic plants can be produced by FLP/FRT site‐specific recombination (Nandy and Srivastava, ; Nanto and Ebinuma, ), Cre/lox site‐specific recombination (Li et al ., ; Mészáros et al ., ; Mlynarova and Nap, ; Moravcikova et al ., ), multi‐auto‐transformation (Khan et al ., ), co‐transformation (Miller et al ., ) and using a marker‐free binary vector (McCormac et al ., ). Among these methods, co‐transformation is the most efficient and simple technique (Tuteja et al ., ).…”