Abstract
Key messageExpression of Cre recombinase by AtRps5apro or AtDD45pro enabled Cre/lox-mediated recombination at an early embryonic developmental stage upon crossing, activating transgenes in the hybrid cowpea and tobacco.Genetic engineering ideally results in precise spatiotemporal control of transgene expression. To activate transgenes exclusively in a hybrid upon fertilization, we evaluated a Cre/lox-mediated gene activation system with the Cre recombinase expressed by either AtRps5a or AtDD45 promoters that showed activity in egg cells and young embryos. In crosses between Cre recombinase lines and transgenic lines harboring a lox-excision reporter cassette with ZsGreen driven by the AtUbq3 promoter after Cre/lox-mediated recombination, we observed complete excision of the lox-flanked intervening DNA sequence between the AtUbq3pro and the ZsGreen coding sequence in F1 progeny upon genotyping but no ZsGreen expression in F1 seeds or seedlings. The incapability to observe ZsGreen fluorescence was attributed to the activity of the AtUbq3pro. Strong ZsGreen expression in F1 seeds was observed after recombination when ZsGreen was driven by the AtUbq10 promoter. Using the AtDD45pro to express Cre resulted in more variation in recombination frequencies between transgenic lines and crosses. Regardless of the promoter used to regulate Cre, mosaic F1 progeny were rare, suggesting gene activation at an early embryo-developmental stage. Observation of ZsGreen-expressing tobacco embryos at the globular stage from crosses with the AtRps5aproCre lines pollinated by the AtUbq3prolox line supported the early activation mode.
Highlights
Precise gene regulation is critical in genetic engineering
The cassette AtUbq3prolox was transferred into the T-DNA region of the binary vector pORE (Coutu et al 2007), together with a kanamycin resistance cassette (S1pronptII) wherein the neomycin phosphotransferase II (nptII) gene containing a Ricinus communis catalase 1 (CAT1) intron was controlled by the S1 promoter and S3 terminator from subterranean clover stunt virus (SCSV) (Schünmann et al 2003) (Table S1)
This study demonstrates the efficacy of Cre/lox-mediated gene activation through genetic crosses in cowpea
Summary
Precise gene regulation is critical in genetic engineering. Conditional transgene activation usually is preferred since ectopic expression of transgenes may result in undesired phenotypes. With the “activator” regulated by a tissue-specific or inducible promoter, a Communicated by Baochun Li. two-component system potentially can lead to tighter control of spatiotemporal transgene expression. The Cre/lox system is one of the most well-characterized two-component systems and has been broadly used in eukaryotic organisms, including plants (Branda and Dymecki 2004; Gilbertson 2003; Srivastava and Thomson 2016). Depending on the numbers of the lox sites employed and their orientations, Cre/lox-mediated recombination can result in excision, inversion, integration, or sequence exchange between two nonhomologous chromosomes (Branda and Dymecki 2004; Srivastava and Thomson 2016).
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