A recombineering‐based gene tagging system for Arabidopsis

Abstract

SUMMARYOne 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.

MYB36 regulates the transition from proliferation to differentiation in the Arabidopsis root

“…The JaTY clone JaTY60A10 (64,154 bp) was used to generate the rMYB36:GFP recombineering line as described (36). The oligos MYB36_Rec_F/R were designed to remove the STOP codon upon recombination with the GFP cassette.…”

Arabidopsis CML38, a Calcium Sensor That Localizes to Ribonucleoprotein Complexes under Hypoxia Stress

“…A similar Student's t test showed no significant difference between wild-type and CML38 plants. generated using a recombineering method (Zhou et al, 2011). The advantage of this approach is that it results in in situ production of a gene encoding the protein of interest tagged with a fluorescent reporter within the larger genomic context, which avoids the exclusion of cis-regulatory elements (Zhou et al, 2011).…”

“…generated using a recombineering method (Zhou et al, 2011). The advantage of this approach is that it results in in situ production of a gene encoding the protein of interest tagged with a fluorescent reporter within the larger genomic context, which avoids the exclusion of cis-regulatory elements (Zhou et al, 2011). To verify that the CML38-YFP recombineering plants respond to hypoxia treatment, plants grown hydroponically were perfused with nitrogen gas and the levels of CML38-YFP transcripts and CML38-YFP protein were analyzed (Fig.…”

“…Recombineering lines containing CML38 fused to three copies of YFP at the C terminus (CML38-YFP) were generated as described (Zhou et al, 2011). The JAtY clone information and primers for CML38 (JAtY53G16) were obtained from resources available at http://www4.ncsu.edu/~jmalonso/AlonsoStepanova_Arabidopsis_localizome.html.…”

“…The cassette was introduced to the C terminus of CML38 by PCR using Rec_F and R primers (Supplemental Table S3). The 3xYpet-tagged JAtY53G16 clone was transformed into A. tumefaciens GV3101, and transgenic recombineering strains were selected by the procedure of Zhou et al (2011) using the 3xYFP_SEQ primers listed in Supplemental Table S3. Growth of the recombineering transgenic plants was done by selection on Murashige and Skoog medium supplemented with 25 mg mL 21 Basta (Chem Service; N12111).…”