RRC ID 51833
Author Kobayashi T, Yagi Y, Nakamura T.
Title Development of Genome Engineering Tools from Plant-Specific PPR Proteins Using Animal Cultured Cells.
Journal Methods Mol Biol
Abstract The pentatricopeptide repeat (PPR) motif is a sequence-specific RNA/DNA-binding module. Elucidation of the RNA/DNA recognition mechanism has enabled engineering of PPR motifs as new RNA/DNA manipulation tools in living cells, including for genome editing. However, the biochemical characteristics of PPR proteins remain unknown, mostly due to the instability and/or unfolding propensities of PPR proteins in heterologous expression systems such as bacteria and yeast. To overcome this issue, we constructed reporter systems using animal cultured cells. The cell-based system has highly attractive features for PPR engineering: robust eukaryotic gene expression; availability of various vectors, reagents, and antibodies; highly efficient DNA delivery ratio (>80 %); and rapid, high-throughput data production. In this chapter, we introduce an example of such reporter systems: a PPR-based sequence-specific translational activation system. The cell-based reporter system can be applied to characterize plant genes of interested and to PPR engineering.
Volume 1469
Pages 147-55
Published 2016-1-1
DOI 10.1007/978-1-4939-4931-1_11
PMID 27557692
MeSH 5' Untranslated Regions Animals Arabidopsis Proteins / genetics* Arabidopsis Proteins / metabolism Binding Sites Carrier Proteins / genetics* Carrier Proteins / metabolism Cells, Cultured Eukaryotic Initiation Factor-4G / genetics Eukaryotic Initiation Factor-4G / metabolism Genetic Engineering / methods* Genetic Vectors HEK293 Cells Humans Recombinant Fusion Proteins / genetics* Recombinant Fusion Proteins / metabolism Repetitive Sequences, Amino Acid Transfection
Times Cited 1
Human and Animal Cells 293T(RCB2202)