A new location to split Cre recombinase for protein fragment complementation.
We have previously described a recombinase-mediated gene stacking system in which the Cre recombinase is used to remove lox-site flanked DNA no longer needed after each round of Bxb1 integrase-mediated site-specific integration. The Cre recombinase can be conveniently introduced by hybridization with a cre-expressing plant. However, maintaining an efficient cre-expressing line over many generations can be a problem, as high production of this DNA-binding protein might interfere with normal chromosome activities. To counter this selection against high Cre activity, we considered a split-cre approach, in which Cre activity is reconstituted after separate parts of Cre are brought into the same genome by hybridization. To insure that the recombinase-mediated gene stacking system retains its freedom to operate, we tested for new locations to split Cre into complementing fragments. In this study, we describe testing four new locations for splitting the Cre recombinase for protein fragment complementation and show that the two fragments of Cre split between Lys244 and Asn245 can reconstitute activity that is comparable to that of wild-type Cre.
Plant Gene Engineering Center, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China.|University of Chinese Academy of Sciences, Beijing, China.
Maryam
M
Rajaee
Plant Gene Engineering Center, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China.
David W
DW
Ow
D017360:Arabidopsis / Q000235:genetics; D018744:DNA, Plant; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D005818:Genetic Engineering / Q000379:methods*; D018745:Genome, Plant; D006824:Hybridization, Genetic; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D020836:Protein Structure, Quaternary; D045522:Recombinases / Q000235:genetics*; D011995:Recombination, Genetic
D018744:DNA, Plant; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D045522:Recombinases; C045073:Cre recombinase; D019426:Integrases
alpha-complementation; gene stacking; protein fragment complementation; site-specific recombination