PVX-Cre-mediated marker gene elimination from transgenic plants.
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 T1 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.
Kopertekh
L
L
Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Virology, Microbiology and Biosafety, Messeweg 11-12, Braunschweig, Germany.
Jüttner
G
G
Schiemann
J
J
D000123:Acetyltransferases / Q000235:genetics; D000613:Aminobutyrates / Q000494:pharmacology; D015139:Blotting, Southern; D018744:DNA, Plant / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D019426:Integrases / Q000235:genetics*; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D017863:Potexvirus / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D014026:Nicotiana / Q000187:drug effects / Q000235:genetics*; D014170:Transformation, Genetic; D014764:Viral Proteins / Q000235:genetics*
D000613:Aminobutyrates; D018744:DNA, Plant; D005819:Genetic Markers; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C003121:phosphinothricin; D000123:Acetyltransferases; C056243:phosphinothricin N-acetyltransferase; C045073:Cre recombinase; D019426:Integrases