Marker-free site-specific gene integration in rice based on the use of two recombination systems.
Transgene 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.
Department of Crop, Soil & Environmental Sciences, University of Arkansas, Fayetteville, AR, USA.
Soumen
S
Nandy
Vibha
V
Srivastava
D001709:Biotechnology / Q000379:methods*; D018556:Crops, Agricultural / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D018390:Gene Targeting / Q000379:methods*; D005819:Genetic Markers; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D014170:Transformation, Genetic; D019076:Transgenes
D005819:Genetic Markers