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A recombineering-based gene tagging system for Arabidopsis

Author:
Zhou, Rongrong, Benavente, Larissa M., Stepanova, Anna N., Alonso, Jose M.
Source:
The plant journal 2011 v.66 no.4 pp. 712-723
ISSN:
0960-7412
Subject:
Arabidopsis, bacterial artificial chromosomes, clones, fluorescent labeling, gene expression, homologous recombination, polymerase chain reaction, recombinant fusion proteins, regulatory sequences, reporter genes, sequence homology
Abstract:
One of the most information-rich aspects of gene functional studies is characterization of gene expression profiles at cellular resolution, and subcellular localization of the corresponding proteins. These studies require visualization of the endogenous gene products using specific antibodies, or, more commonly, generation of whole-gene translational fusions with a reporter gene such as a fluorescent protein. To facilitate the generation of such translational fusions and to ensure that all cis-regulatory sequences are included, we have used a bacterial homologous recombination system (recombineering) to insert fluorescent protein tags into genes of interest harbored by transformation-competent bacterial artificial chromosomes (TACs). This approach has several advantages compared to other classical strategies. First, the researcher does not have to guess what the regulatory sequences of a gene are, as tens of thousands of base pairs flanking the gene of interest can be included in the construct. Second, because the genes of interest are not amplified by PCR, there are practically no limits to the size of a gene that can be tagged. Third, there are no restrictions on the location in which the fluorescent protein can be inserted, as the position is determined by sequence homology with the recombination primers. Finally, all of the required strains and TAC clones are publically available, and the experimental procedures described here are simple and robust. Thus, we suggest that recombineering-based gene tagging should be the gold standard for gene expression studies in Arabidopsis.
Agid:
479583