Evaluation of CRE-mediated excision approaches in Arabidopsis thaliana.
The ability of the CRE recombinase to catalyze excision of a DNA fragment flanked by directly repeated lox sites has been exploited to modify gene expression and proved to function well in particular case studies. However, very often variability in CRE expression and differences in efficiency of CRE-mediated recombination are observed. Here, various approaches were investigated to reproducibly obtain optimal CRE activity. CRE recombination was analyzed either by transforming the CRE T-DNA into plants containing a lox-flanked fragment or by transforming a T-DNA harboring a lox-flanked fragment into plants producing the CRE recombinase. Although somatic CRE-mediated excision of a lox-flanked fragment was obtained in all transformants, a variable amount of germline-transmitted deletions was found among different independent transformants, irrespective of the orientation of transformation. Also, the efficiency of CRE-mediated excision correlated well with the CRE mRNA level. In addition, CRE-mediated fragment excision was compared after floral dip and after root tissue transformation when transforming in a CRE-expressing background. Importantly, less CRE activity was needed to excise the lox-flanked fragment from the transferred T-DNA after root tissue transformation than after floral dip transformation. We hypothesize that this is correlated with the lower T-DNA copy number inserted during root transformation as compared to floral dip transformation.
Marjanac
Gordana
G
Department of Plant Systems Biology, VIB, Technologiepark 927, Gent 9052, Belgium.
De Paepe
Annelies
A
Peck
Ingrid
I
Jacobs
Anni
A
De Buck
Sylvie
S
Depicker
Anna
A
D017360:Arabidopsis / Q000235:genetics* / Q000254:growth & development; D004269:DNA, Bacterial / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D018745:Genome, Plant; D005966:Glucuronidase / Q000378:metabolism; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D018517:Plant Roots / Q000235:genetics* / Q000254:growth & development; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000254:growth & development; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014170:Transformation, Genetic
D004269:DNA, Bacterial; D018744:DNA, Plant; D012333:RNA, Messenger; C036483:T-DNA; C045073:Cre recombinase; D019426:Integrases; D005966:Glucuronidase