The alc-GR system: a modified alc gene switch designed for use in plant tissue culture.
The ALCR/alcA (alc) two-component, ethanol-inducible gene expression system provides stringent control of transgene expression in genetically modified plants. ALCR is an ethanol-activated transcription factor that can drive expression from the ALCR-responsive promoter (alcA). However, the alc system has been shown to have constitutive expression when used in plant callus or cell suspension cultures, possibly resulting from endogenous inducer produced in response to lowered oxygen availability. To widen the use of the alc system in plant cell culture conditions, the receptor domain of the rat glucocorticoid receptor (GR) was translationally fused to the C terminus of ALCR to produce ALCR-GR, which forms the basis of a glucocorticoid-inducible system (alc-GR). The alc-GR switch system was tested in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells using a constitutively expressed ALCR-GR with four alternative alcA promoter-driven reporter genes: beta-glucuronidase, endoplasmic reticulum-targeted green fluorescent protein, haemagglutinin, and green fluorescent protein-tagged Arabidopsis (Arabidopsis thaliana) Arath;CDKA;1 cyclin-dependent kinase. Gene expression was shown to be stringently dependent on the synthetic glucocorticoid dexamethasone and, in cell suspensions, no longer required ethanol for induction. Thus, the alc-GR system allows tight control of alcA-driven genes in cell culture and complements the conventional ethanol switch used in whole plants.
Roberts
Gethin R
GR
Department of Cell and Developmental Biology, John Innes Centre, Norwich NR4 7UH, United Kingdom.
Garoosi
G Ali
GA
Koroleva
Olga
O
Ito
Masaki
M
Laufs
Patrick
P
Leader
David J
DJ
Caddick
Mark X
MX
Doonan
John H
JH
Tomsett
A Brian
AB
D000818:Animals; D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D000431:Ethanol / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant; D005812:Genes, Switch; D049452:Green Fluorescent Proteins / Q000235:genetics; D018551:Solanum lycopersicum / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014026:Nicotiana / Q000235:genetics*; D014157:Transcription Factors
C448681:ALCATRAZ protein, Arabidopsis; D029681:Arabidopsis Proteins; D017931:DNA Primers; D010940:Plant Proteins; D018749:RNA, Plant; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D000431:Ethanol