Expression of a transgene exchanged by the recombinase-mediated cassette exchange (RMCE) method in plants

Abstract

We developed a site-directed integration (SDI) system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined target locus by recombinase-mediated cassette exchange (RMCE). We produced site-specific transgenic tobacco plants from four target lines and examined expression of the transgene in T1 site-specific transgenic tobacco plants, which were obtained by backcrossing. We found that site-specific transgenic plants from the same target lines showed approximately the same level of expression of the transgene. Moreover, we demonstrated that site-specific transgenic plants showed much less variability of transgene expression than random-integration transgenic plants. Interestingly, transgenes in the same direction at the same target locus showed the same level of activity, but transgenes in different directions showed different levels of activity. The expression levels of transgene did not correlate with those of the target gene. Our results showed that the SDI system could benefit the precise comparisons between different gene constructs, the characterization of different chromosomal regions and the cost-effective screening of reliable transgenic plants.

Approaches for gene targeting and targeted gene expression in plants

“…95,96 This has been tested in several plants including tobacco, Arabidopsis and soybean. 18,87,88,90,94 A combination of MAT vector system and the site-directed integration (SDI) system has been tested in Nicotiana tabacum in order to remove the marker after site-specific integration, producing marker free site-specific transgenic plants. 89 Site-specific transgene switching.…”

“…The production of marker-free transgenic plants has been already reported using site-specific recombination systems in plants. 5,[74][75][76][77][78] In recent years, with the aid of a site-specific recombination system, a sitespecific integration method to insert a desirable gene into a predefined site [79][80][81][82][83][84][85][86] and the recombinase-mediated cassette exchange (RMCE) method to exchange a desirable gene with a target gene at a predefined site [87][88][89][90] are being developed 91 and will be discussed briefly (see below).…”

Recombinase technology: applications and possibilities

“…Removal of the selectable marker also allows reuse of the same selection regime for subsequent rounds of gene transfer. A number of recombinase-mediated marker deletion strategies have been reported in model plants (Dale and Ow 1991; Albert et al 1995; Gleave et al 1999; Sugita et al 1999, 2000a, b; Endo et al 2001, 2002; Hohn et al 2001; Hare and Chua 2002; Nanto et al 2005; Nanto and Ebinuma 2008; Nanto et al 2009; Thomson et al 2009, 2010), as well as in crop species (Lyznik et al 1996; Srivastava et al 1999; Ebinuma and Komamine 2001; Hoa et al 2002; Matsunaga et al 2002; Gilbertson et al 2003; Srivastava and Ow 2003; Zhang et al 2003; Kerbach et al 2005; Radhakrishnan and Srivastava 2005; Sreekala et al 2005; Ballester et al 2006; Cao et al 2006; Chawla et al 2006; Cuellar et al 2006; Djukanovic et al 2008; Hu et al 2008; Kempe et al 2010).

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“…This technology has allowed the production of precisely engineered transgenic plants and has been reported to function in Arabidopsis (Louwerse et al 2007; Vergunst and Hooykaas 1998; Vergunst et al 1998), aspen (Fladung and Becker 2010), tobacco (Albert et al 1995; Choi et al 2000; Day et al 2000; Nanto et al 2005, 2009; Nanto and Ebinuma 2008) maize (Baszczynski et al 2003; Kerbach et al 2005), rice (Srivastava and Ow 2002; Srivastava et al 2004; Chawla et al 2006), soybean (Li et al 2009) and the plastid genome of tobacco (Lutz et al 2004). Rates of integration have been documented from ~33% in tobacco (Albert et al 1995; Day et al 2000) to nearly 50% in rice (Srivastava et al 2004).…”

Directed genome engineering for genome optimization

“…DSB-mediated recombination allows removal of both transgene and endogene sequences, whereas site-specific recombination has been limited to transgene sequences preconfigured with recombination sites (Nanto et al, 2009). An important application of both technologies for crop improvement is the removal of selectable marker genes only needed for transformation (Petolino et al, 2010, Zhang et al, 2003.…”