PVX-Cre-mediated marker gene elimination from transgenic plants

Abstract

Cre recombinase gene from bacteriophage P1 was transiently expressed by a Potato Virus X (PVX)-based vector in transgenic lox-target Nicotiana benthamiana plants to remove the selectable marker gene. The target construct consisted of two directly oriented lox sites flanking a bar gene located between a gfp coding region and an upstream CaMV 35S promoter. The Cre-mediated excision of intervening sequence placed the gfp coding region under the transcriptional control of the CaMV 35S promoter. GFP activity was observed in PVX-Cre systemically infected leaves, regenerants from PVX-Cre infected explants and T 1 progeny of these regenerants. PVX-Cre was removed efficiently from the regenerants by adding the nucleoside analogue ribavirin to the culture medium. Molecular data proved a correlation between gfp expression and precise site-specific excision of the bar gene in all examined transgenic lines. The frequency of recombination expressed as a percentage of regenerated plants exhibiting marker gene excision varied from 48% to 82%. These results demonstrate that a plant virus vector can be used efficiently to express cre recombinase in vivo providing an alternative method for the production of transgenic plants without marker genes.

Advances in genetic engineering of marine algae

“…The selection marker removal may significantly reduce the public acceptance of genetically modified plants (Miki and McHugh, 2004). Several marker elimination methods in higher plants have been developed during these years including co-transformation, which is usually applied in Agrobacterium-mediated transformation (Sripriya et al, 2008) and various site-specific recombination methods that eliminate the selection marker by deleting or inverting the marker gene with the help of an enzyme recombinase (Cotsaftis et al, 2002;Darbani et al, 2007, Kopertekh et al, 2004, Ow, 2002. The marine algal genetic transformation will certainly meet these biosafety issues (more discussion can be found in Section 6) when cultivating transgenic microalgae in an open pond of a coastal area or transgenic macroalgal sporophytes in the open sea.…”

Elimination of Transgenic Sequences in Plants by Cre Gene Expression

“…Two Cre-virus vectors, PVX-Cre (Kopertekh et al, 2004a(Kopertekh et al, , 2004b and TMV-Cre (Jia et al, 2006), have been shown to be functional in loxtarget N. benthamiana and N. tabacum plants. In both vectors the cre gene was integrated between movement and coat protein genes.…”

“…Another option to perform transient cre expression is the use of Cre-virus vectors. The first plant Cre-virus vector was based on PVX and demonstrated high recombination rates (48-82%) in N. benthamiana (Kopertekh et al, 2004b). This vector was also suitable to generate marker-free tobacco plants without a regeneration step (Kopertekh et al, 2004a).…”

Generating Marker-Free Transgenic Wheat Using Minimal Gene Cassette and Cold-Inducible Cre/Lox System

“…The most frequently used recombination approach in transgenic plants is the Cre/lox system. Recombination events have also been achieved in animal and plant systems (Kopertekh et al 2004).…”