Expression of epitope-tagged proteins in Arabidopsis leaf mesophyll protoplasts.
Advances in genomic and proteomic platforms enable high-throughput studies for regulatory factors and interactors involved in signaling network at a molecular level. However, it has never been trivial to verify the omics data in vivo or functionally integrate the data in a cell signaling context. For plants, genetic approaches using knockout mutants and transgenic lines have been mainly used to characterize functions of gene products in vivo. In general, such approaches demand a longer time and a higher cost and have difficulties in understanding gene functions comprehensively in a high-throughput manner. Transient gene expression is a method of choice to examine the cellular functions of genetic components in vivo. The leaf mesophyll protoplasts (LMP) provide a perfect system to transiently express a gene encoding an epitope-tagged protein of interest and quickly and reliably trace subcellular locales of the protein in a near high-throughput manner. Here, a simple and straightforward method for isolating leaf meshophyll protoplasts from Arabidopsis has been described in detail to help beginners initiate their first cell-based functional genomic analysis.
Cho
Young-Hee
YH
Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA.
Yoo
Sang-Dong
SD
D017360:Arabidopsis / Q000235:genetics*; D000939:Epitopes / Q000235:genetics* / Q000276:immunology; D015870:Gene Expression; D018515:Plant Leaves / Q000166:cytology* / Q000235:genetics*; D011523:Protoplasts / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology
D000939:Epitopes; D011993:Recombinant Fusion Proteins