Marker‐free site‐specific gene integration in rice based on the use of two recombination systems

Abstract

SummaryTransgene integration mediated by heterologous site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. This approach of plant transformation generates a precise site-specific integration (SSI) structure consisting of a single copy of the transgene construct. As a result, stable transgene expression correlated with promoter strength and gene copy number is observed among independent transgenic lines and faithfully transmitted through subsequent generations. Site-specific integration approaches use selectable marker genes, removal of which is necessary for the implementation of this approach as a biotechnology application. As SSR systems are also excellent tools for excising marker genes from transgene locus, a molecular strategy involving gene integration followed by marker excision, each mediated by a distinct recombination system, was earlier proposed. Experimental validation of this approach is the focus of this work. Using FLPe-FRT system for site-specific gene integration and heat-inducible Cre-lox for marker gene excision, marker-free SSI lines were developed in the first generation itself. More importantly, progeny derived from these lines inherited the marker-free locus, indicating efficient germinal transmission. Finally, as the transgene expression from SSI locus was not altered upon marker excision, this method is suitable for streamlining the production of marker-free SSI lines.

Strong activity of FLPe recombinase in rice plants does not correlate with the transmission of the recombined locus to the progeny

“…Consistent with its activity in the germline, the ZmUBI1:Cre construct successfully generated stable marker-free lines of rice and wheat (Moore and Srivastava 2006;Srivastava et al 1999). Similarly, other promoters, including the 35S promoter, heat-inducible, and chemical-inducible promoters, have also been successful in generating stable marker-free lines when the Cre-lox system was used for marker excision (Bala et al 2013;Dale and Ow 1991;Nandy and Srivastava 2012;Russell et al 1992;Sreekala et al 2005;Zhang et al 2003). Thus, gametophytic promoters have mostly been used for pollen-specific excision of the transgene for biocontainment applications (Luo et al 2007;Mlynárová et al 2006).…”

Gene stacking by recombinases

“…Integration of full‐length DNA constructs whether containing a single gene or a multigene cassette is the most important criteria in the stability of gene expression from SSI locus as each transcription unit remains intact, and aberrant transcription is avoided. Additionally, gene dosage‐dependent expression has been observed from SSI lines containing allelic gene copies or from an array of genes in the chromosome (Akbudak et al ., ; Chawla et al ., ; Nandy and Srivastava, ). The presence of random integrations, as expected, frequently causes gene silencing in SSI line, which is mostly reversed upon their segregation of the unwanted gene fragments in the progeny (Chawla et al ., ).…”

“…Strategies of marker-free site-specific integration (MF-SSI) were designed long ago (Darbani et al, 2007;Fladung and Becker, 2010;Srivastava and Ow, 2004;Wang et al, 2011), but their experimental demonstration awaited the development of new SSR systems (Figure 4). A combination of Cre-lox and R-RS or FLP-FRT or phiC31 or Bxb1 has been successfully used for developing marker-free SSI lines of tobacco or rice (De Paepe et al, 2013;Hou et al, 2014;Nandy and Srivastava, 2012;Nanto and Ebinuma, 2008). In these studies, Cre-lox was dedicated to SMG excision due to its efficiency in allowing the recovery of marker-free lines without applying selection pressure.…”

High efficiency Agrobacterium‐mediated site‐specific gene integration in maize utilizing the FLP‐FRT recombination system

“…In the presence of a recombinase, the donor DNA is exchanged with the target, a process referred to as recombinase‐mediated cassette exchange (RMCE). This approach has been applied to transgene insertion in plants (Ebinuma et al ., ; Li et al ., ; Louwerse et al ., ; Nandy and Srivastava, , ; Nanto and Ebinuma, ; Nanto et al ., , ; Srivastava and Thomson, ).…”