Homologous recombination-mediated knock-in targeting of the MET1a gene for a maintenance DNA methyltransferase reproducibly reveals dosage-dependent spatiotemporal gene expression in rice |
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Authors: | Takaki Yamauchi Yasuyo Johzuka-Hisatomi Sachiko Fukada-Tanaka Rie Terada Ikuo Nakamura Shigeru Iida |
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Affiliation: | National Institute for Basic Biology, Okazaki 444-8585, Japan;, Graduate School of Science and Technology, Chiba University, Matsudo 271-8510, Japan;, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka 422-8526, Japan;, Graduate School of Horticulture, Chiba University, Matsudo 271-8510, Japan;, and School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan |
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Abstract: | Although homologous recombination-promoted knock-in targeting to monitor the expression of a gene by fusing a reporter gene with its promoter is routine practice in mice, gene targeting to modify endogenous genes in flowering plants remains in its infancy. In the knock-in targeting, the junction sequence between a reporter gene and an endogenous target promoter can be designed properly, and transgenic plants carrying an identical and desired knock-in allele can be repeatedly obtained. By employing a reproducible gene-targeting procedure with positive–negative selection in rice, we were able to obtain fertile transgenic knock-in plants with the promoterless GUS reporter gene encoding β-glucuronidase fused with the endogenous promoter of MET1a , one of two rice MET1 genes encoding a maintenance DNA methyltransferase. All of the primary (T0) transgenic knock-in plants obtained were found to carry only one copy of GUS , with the anticipated structure in the heterozygous condition, and no ectopic events associated with gene targeting could be detected. We showed the reproducible, dosage-dependent and spatiotemporal expression of GUS in the selfed progenies of independently isolated knock-in targeted plants. The results in knock-in targeted plants contrast sharply with the results in transgenic plants with the MET1a promoter -fused GUS reporter gene integrated randomly in the genome: clear interindividual variation of GUS expression was observed among independently obtained plants bearing the randomly integrated transgenes. As our homologous recombination-mediated gene-targeting strategy with positive–negative selection is, in principle, applicable to modify any endogenous gene, knock-in targeting would facilitate basic and applied plant research. |
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Keywords: | knock-in gene targeting promoter activity GUS reporter gene dosage-dependent expression rice MET1 gene |
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