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Conditional, recombinase‐mediated expression of genes in plant cell cultures
Author(s) -
Joubès Jérôme,
De Schutter Kristof,
Verkest Aurine,
Inzé Dirk,
De Veylder Lieven
Publication year - 2004
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2004.02004.x
Subject(s) - biology , cre recombinase , transgene , gene , microbiology and biotechnology , genetics , genetically modified mouse
Summary In plant cells, overexpression of critical genes can be hampered by deleterious effects on development that results in a counterselection of transgenic cells harboring the gene of interest. Inducible expression systems have been reported, but many of them show unwanted leaky expression. To circumvent this potential problem, a novel inducible system was developed based on two previously characterized systems: the CRE‐ loxP site‐specific recombination system of bacteriophage P1 and the subcellular targeting of proteins by a mammalian glucocorticoid receptor (GR). By fusing the receptor domain of the rat GR to the carboxyl terminus of the CRE recombinase, a double‐lock conditional transcriptional induction system was created that is highly useful to overexpress genes whose expression may block transgenic regeneration. Furthermore, because the designed vector utilizes the GATEWAY™ recombination technology, cloning was restriction‐ and ligation‐free, thus rendering the vector suitable for high‐throughput research. The system was tested in Nicotiana tabacum bright yellow‐2 (BY‐2) cells and its efficiency was demonstrated for the controlled overexpression of the gus reporter gene and a mutant allele of the A‐type cyclin‐dependent kinase (CDKA), which is known to be a potent inhibitor of the cell cycle.