Dexamethasone-Inducible Green Fluorescent Protein Gene Expression in Transgenic Plant Cells

Genomic research has made a large number of sequences of novel genes orexpressed sequence tags available. To investigate functions of these genes, a system for conditionalcontrol of gene expression would be a useful tool. Inducible trans-gene expression that uses greenfluorescent protein gene (gfp) as a reporter gene has been investigated in transgenic cell lines ofcotton (COT; Gossypium hirsutum L.), Fraser fir [FRA; Abies fraseri (Pursh) Poir], Nordmann fir(NOR; Abies nord-manniana Lk.), and rice (RIC; Oryza sativa L. cv. Radon). Transgenic cell lineswere used to test the function of the chemical inducer dexamethasone. Inducible transgene expressionwas observed with fluorescence and confocal microscopy, and was confirmed by northern blotanalyses. Dexamethasone at 5 mg/L induced gfp expression to the nearly highest level 48 h aftertreatment in COT, FRA, NOR, and RIC. Dexamethasone at 10 mg/L inhibited the growth of transgeniccells in FRA and NOR, but not COT and RIC. These results demonstrated that concentrations of inducerfor optimum inducible gene expression system varied among transgenic cell lines. The inducible geneexpression system described here was very effective and could be valuable in evaluating thefunction of novel gene.

A New β-Estradiol-lnducible Vector Set that Facilitates Easy Construction and Efficient Expression of Transgenes Reveals CBL3-Dependent Cytoplasm to Tonoplast Translocation of CIPK5

Transient and stable expression of transgenes is central to many investigations in plant biology research. Chemical regulation of expression can circumvent problems of plant lethality caused by constitutive overexpression or allow inducible knock （out/down） approaches. Several chemically inducible or repressible systems have been described and successfully applied. However, cloning and application-specific modification of most available inducible expression systems have been limited and remained complicated due to restricted cloning options. Here we describe a new set of 57 vectors that enable transgene expression in transiently or stably transformed cells. All vectors harbor a synthetically optimized XVE expression cassette, allowing I^-estradiol mediated protein expression. Plasmids are equipped with the reporter genes GUS, GFP, mCherry, or with HA and Strepll epitope tags and harbor an optimized multiple cloning site for flexible and simple clon- ing strategies. Moreover, the vector design allows simple substitution of the driving promoter to achieve tissue-specificity or to modulate expression ranges of inducible transgene expression. We report details of the kinetics and dose-dependence of expression induction in Arabidopsis leaf mesophyll protoplasts, transiently transformed Nicotiana benthamiana leaves, and stably transformed Arabidopsis plants. Using these vectors, we investigated the influence of CBL （Calcineurin B-like） protein expression on the subcellular localization of CIPKs （Calcineurin B-like interacting protein kinases）. These analyses uncovered that induced co-expression of CBL3 is fully sufficient for dynamic translocation of CIPK5 from the cytoplasm to the tonoplast. Thus, the vector system presented here facilitates a broad range of research applications.

An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase

Inducible gene-expression systems play important roles in gene functional assays in the post-genome era.Streptomyces phage-derived phiC31 integrase,which mediates an irreversible site-specific cassette exchange between the phage attachment site(attP)and the bacterial attachment site(attB),provides a promising option for the construction of a controllable gene-expression system.Here,we report a phiC3I integrase-mediated promoter flip system(FLIP)for the inducible expression of target genes in silk-worm(Bombyx mori).First,we constructed a FLIP reporter system,in which a BmAct4 promoter with enhanced translational efficiency was flanked by the attB and attP sites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter gene.The coexpression of a C-terminal modified phiC3 I-NLS integrase carrying a simian virus 40(SV40)nuclear localization signal(NLS)effectively flipped the BmAct4 promoter through an attBlattP exchange,thereby activating the downstream expression of DsRed in a silkworm embryo-derived cell line,BmE.Subsequently,the FLIP system,together with a system continuously expressing the phiC3 I-NLS integrase,was used to construct binary transgenic silkworm lines.Hybridization between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter,with an approximately 39%heritable transformation efficiency in silkworm offispring,leading to the constitutive and high-level expression of DsRed in silkworms,which accounted for approximately 0.81%of the silkworm pupal weight.Our successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.