<Emphasis Type="Italic">Renilla</Emphasis> luciferase-<Emphasis Type="Italic">Aequorea</Emphasis> GFP (Ruc-GFP) fusion protein,a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals

Light-emitting reporter proteins play an increasing role in the study of gene expression in vitro and in vivo. Here we present a ruc-gfp fusion gene construct generated by fusing a cDNA for Renilla luciferase (ruc) in-frame with a cDNA encoding the "humanized" GFP (gfp) from Aequorea. A plasmid containing the fusion gene construct was successfully transformed into, and expressed in, mammalian cells. The transformed cells exhibited both Renilla luciferase activity in the presence of coelenterazine and GFP fluorescence upon excitation with UV light. Spectrofluorometry of cells containing the Ruc-GFP fusion protein, in the absence of wavelengths capable of exciting GFP fluorescence but in the presence of the luciferase substrate, coelenterazine, showed an emission spectrum with two peaks at 475 nm and 508 nm. These two peaks correspond to the emission maximum of Renilla luciferase at 475 nm and that of GFP at 508 nm. The peak at 508 nm generated in the presence of coelenterazine alone (without UV excitation) is the result of intramolecular energy transfer from Renilla luciferase to Aequorea GFP. Southern analysis of genomic DNA purified from transformed Chinese hamster ovary (CHO) cells and fluorescence in situ hybridization (FISH) to metaphase chromosomes confirmed the integration of the ruc-gfp fusion gene on a single chromosome. The bifunctional Ruc-GFP fusion protein allows the detection of gene expression at the single-cell level based on green fluorescence, and in a group of cells based on luminescence emission. Furthermore, animal experiments revealed that light emission from the Ruc-GFP fusion protein can be detected externally in the organs or tissues of live animals bearing the gene construct.     

Construction and testing of an intron-containing luciferase reporter gene from<Emphasis Type="Italic">Renilla reniformis</Emphasis>

We describe a newRenilla reniformis luciferase reporter gene,RiLUC, which was designed to allow detection of luciferase activity in studies involvingAgrobacterium-based transient expression studies. TheRLUC gene was altered to contain a modified intron from the castor bean catalase gene while maintaining consensus eukaryotic splicing sites recognized by the plant spliceosome.RLUC andRiLUC reporter genes were fused to the synthetic plant SUPER promoter. Luciferase activity within agrobacteria containing the SUPER-RLUC construct increased during growth in culture. In contrast, agrobacteria harboring the SUPER-RiLUC gene fusion showed no detectable luciferase activity. Agrobacteria containing these gene fusions were cotransformed with a compatible normalization plasmid containing a cauliflower mosaic virus 35S promoter (CaMV) joined to the firefly luciferase coding region (FiLUC) and infused into tobacco leaf tissues through stomatal openings. The kinetics of luciferase production from theRLUC orRiLUC reporters were consistent, with expression of theRiLUC gene being limited to transiently transformed plant cells.RiLUC activity from the reporter gene fusions was measured transiently and within stably transformed tobacco leaf tissues. Analysis of stably transformed tobacco plants harboring either reporter gene fusion showed that the intron altered neither the levels of luciferase activity nor tissue-specific expression patterns driven by the SUPER promoter. These results demonstrate that theRiLUC reporter gene can be used to monitor luciferase expression in transient and stable transformation experiments without interference from contaminating agrobacteria.     

The firefly luciferase gene as a non-invasive reporter for Dendrobium transformation

Here a screening method is described for transformed tissues and transgenic plants of Dendrobium (Orchidaceae) using the firefly luciferase gene ( luc ) as a combined marker/reporter gene. Protocorm-likebodies (PLB) were bombarded with tungsten particles (1.3 µm) coated with plasmids carrying a 35S-luc chimeric gene. Three weeks after bombardment 1 mM luciferin was added to the tissues and transformed cells were identified by virtue of their bioluminescence as monitored by low-light video microscopy in combination with a real-time photon imaging technique. Transformed tissues were excised, allowed to proliferate, and then subjected to a second round of screening. After three rounds of growth and screening, transformed Dendrobium tissues expressing luciferase were used to generate transgenic plants. Southern blot analysis of several transgenic lines confirmed the integration of the luciferase gene into the orchid genome. It is thought that this procedure can be used for transformation of not only orchids but other species as well.     

Infection of potato mesophyll protoplasts with five plant viruses

Methods are described for preparing potato mesophyll protoplasts that are suitable for infection with inocula of virus nucleoprotein or RNA. The protoplasts could be infected with four sap-transmissible viruses (tobacco mosaic, tobacco rattle, tobacco ringspot and tomato black ring viruses) and with potato leafroll virus, which is not saptransmissible. No differences were observed in ability to infect protoplasts with potato leafroll virus strains differing either in virulence in intact plants or in aphid transmissibility.     

Bioluminescent detection of endotoxin effects on HIV-1 LTR-driven transcription in vivo.

We investigated the effects of Gram-negative bacterial lipopolysaccharide (LPS) on luciferase expression in transgenic reporter mice in which luciferase expression is driven by the nuclear factor kappaB (NF-kappaB)-dependent portion of the human immunodeficiency virus-1 (HIV-1) long terminal repeat (HIV-1 LTR). Using these mice, we dissected the sources of luciferase activity at the organ level by (a) assessing luciferase activity in organ homogenates, (b) bioluminescence imaging in vivo, and (c) bioluminescence imaging of individual organs ex vivo. Luciferin dosage was a critical determinant of the magnitude of photon emission from these reporter mice. Photon emission increased at doses from 0.5-6 mg of luciferin given by intraperitoneal (IP) injection. The differential between basal and LPS-induced bioluminescence was maximal at 3-6 mg of luciferin. Luciferase expression was highly inducible in lungs, liver, spleen, and kidneys after a single IP injection of LPS, as assessed by luciferase activity measurements in organ homogenates. Luciferase activity was also induced in the forebrain by treatment with IP LPS. In contrast, aerosolized LPS produced a response localized to the lungs as assessed by both bioluminescence and ex vivo luciferase assay measurements. These studies demonstrate the utility of luciferase reporter mice for determining organ-specific gene expression in response to local and systemic stimuli.     

Thein vivo pattern of firefly luciferase expression in transgenic plants

Expression of the firefly luciferase gene in transgenic plants produces light emission patterns when the plants are supplied with luciferin. We explored whether inin vivo pattern of light emission truly reveals the pattern of luciferase gene expression or whether it reflects other parameters such as the availability of the substrate, luciferin, or the tissue-specific distribution of organelles in which luciferase was localized. The tissue-specific distribution of luciferase activity and thein vivo pattern of light were examined when the luciferase gene was driven by different promoters and when luciferase was redirected from the peroxisome, where it is normally targeted, to the chloroplast compartment. It was found that the distribution of luciferase activity closely correlated with the tissue-specific pattern of luciferase mRNA. However, thein vivo light pattern appeared to reflect not only tissue-specific distribution of luciferase activity, but also the pattern of luciferin uptake.     

Luciferase as a reporter gene for transformation studies in rice (Oryza sativa L.)

Transformed rice plants of var `TN1' were regenerated from immature embryos following particle bombardment with a construct containing the firefly luciferase gene as a reporter gene and the hygromycin resistance gene as a selectable marker. Expression of the luciferase gene in the presence of the substrate luciferin was visualised in the calli derived from bombarded immature embryos and in the leaves and roots of the regenerated transformed plants using a low light imaging system (luminograph). Embryogenic callus proliferation and plant regeneration were unaffected by luciferin treatment and luminograph screening. The quantitative Luc assay using samples of leaf tissue from the segregating generations gave early information about the homozygous and hemizygous state of the luc transgene. Received: 25 August 1998 / Revision received: 2 November 1998 / Accepted: 13 November 1998     

Overproduction of alfalfa glutamine synthetase in transgenic tobacco plants

Summary We have obtained transgenic tobacco plants overexpressing the enzyme glutamine synthetase (GS) by fusing an alfalfa GS gene to the cauliflower mosaic virus 35S promotor and integrating it intoNicotiana tabacum var. W38 plants byAgrobacterium tumefaciens mediated gene transfer. The amount of RNA specific to alfalfa GS was about 10 times higher in transgenic tobacco plants than in alfalfa. The alfalfa GS produced by these transgenic plants was identified by Western blotting and represented 5% of total soluble protein in the transformed plants, amounting to a 5-fold increase in specific GS activity and in a 20-fold increase in resistance to the GS inhibitorl-phosphinothricin in vitro. Tissue from GS overproducing plants showed a sevenfold lower amount of free NH₃. The amino acid composition of the plant tissue was not altered significantly by GS overproduction. GS overproducing plants were fertile and grew normally. These data show that a high level of expression of a key metabolic enzyme such as glutamine synthetase does not interfere with growth and fertility of plants.     

Enhanced luciferin entry causes rapid wound-induced light emission in plants expressing high levels of luciferase

In-vivo imaging of transgenic tobacco plants (Nicotiana tobacum L.) expressing firefly luciferase under the control of the Arabidopsis phenylalanine ammonia-lyase 1 (PAL1)-promoter showed that luciferase-catalyzed light emission began immediately after the substrate luciferin was sprayed onto the leaves and reached a plateau phase after approximately 60 min. This luminescence could easily be detected for up to 24 h after luciferin application although the light intensity declined continuously during this period. A strong and rapid increase in light emission was observed within the first minutes after wounding of luciferin-sprayed leaves. However, these data did not correlate with luciferase activity analysed by an in-vitro enzyme assay. In addition, Arabidopsis plants expressing luciferase under the control of the constitutive 35S-promoter showed similar wound-induced light emission. In experiments in which only parts of the leaves were sprayed with luciferin solutions, it was shown that increased uptake of luciferin at the wound site and its transport through vascular tissue were the main reasons for the rapid burst of light produced by preformed luciferase activity. These data demonstrate that there are barriers that restrict luciferin entry into adult plants, and that luciferin availability can be a limiting factor in non-invasive luciferase assays. Received: 11 March 2000 / Accepted: 16 May 2000     

Chimeric vector construction for higher-plant transformation

A chimeric vector pKR612B1 was developed containing the neomycin phosphotransferase (APH) gene from the Tn5 transposon under the control of the gene VI promoter of cauliflower mosaic virus (CaMV), and was used to transform higher plant protoplasts. Plasmid pDOB612, the parental vector of pKR612B1, has two unique restriction sites, SmaI and BamHI, positioned just downstream of the CaMV gene VI promoter sequence. These unique cloning sites can be used for any kind of gene insertion into this vector. Using the polyethylene glycol transformation procedure, a large number of turnip and tobacco protoplasts were transformed and proved to be resistant to kanamycin (Km). From tobacco protoplasts whole Km-resistant plants were regenerated and shown to contain the integrated foreign gene. APH activity was detected in both transformed calli and in regenerated plants. DNA from transformed clones was analysed by Southern blot hybridization, showing the presence of the Tn5-derived gene.     

Non-destructive detection of firefly luciferase (LUC) activity in single plant cells using a cooled, slow-scan CCD camera and an optimized assay

A flexible, comparatively inexpensive system based on a liquid nitrogen-cooled slow-scan CCD (charge coupled device) camera is presented, which can be employed for quantitative low-light (bioluminescence, chemiluminescence or fluorescence) imaging. Using this camera system and the firefly luciferase (LUC) as a screenable marker, transgenic tobacco lines have been produced by direct gene transfer. Bioluminescence emitted from single tobacco cells transiently expressing the firefly luciferase gene (Luc) as well as from stably transformed calli, regenerated shoots, plantlets and T₁ seedlings could be monitored in vivo with no effect on the viability of the material analysed. The patterns of light emission from sections through Luc -expressing leaves and bioluminescent single protoplasts isolated from such leaves were also imaged microscopically. The assay used to detect in vivo LUC activity was optimized by quantifying bioluminescence emitted from Luc -expressing tobacco protoplasts and leaves incubated in different substrate solutions and determining the kinetics of light emission during incubation in the substrate solution.     

Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase

Ascorbate, or vitamin C, is obtained by humans mostly from plant sources. Various approaches have been made to increase ascorbate in plants by transgenic means. Most of these attempts have involved leaf material from model plants, with little success reported using genes from the generally accepted l-galactose pathway of ascorbate biosynthesis. We focused on increasing ascorbate in commercially significant edible plant organs using a gene, GDP-l-galactose phosphorylase (GGP or VTC2), that we had previously shown to increase ascorbate concentration in tobacco and Arabidopsis thaliana. The coding sequence of Actinidia chinensis GGP, under the control of the 35S promoter, was expressed in tomato and strawberry. Potato was transformed with potato or Arabidopsis GGP genes under the control of the 35S promoter or a polyubiquitin promoter (potato only). Five lines of tomato, up to nine lines of potato, and eight lines of strawberry were regenerated for each construct. Three lines of tomato had a threefold to sixfold increase in fruit ascorbate, and all lines of strawberry showed a twofold increase. All but one line of each potato construct also showed an increase in tuber ascorbate of up to threefold. Interestingly, in tomato fruit, increased ascorbate was associated with loss of seed and the jelly of locular tissue surrounding the seed which was not seen in strawberry. In both strawberry and tomato, an increase in polyphenolic content was associated with increased ascorbate. These results show that GGP can be used to raise significantly ascorbate concentration in commercially significant edible crops.     

Improved assay sensitivity of an engineered secreted Renilla luciferase.

We have previously reported the construction of a functional Renilla luciferase enzyme secreted by mammalian cells when fused to the signal peptide of human interleukin-2. The presence of three predicted cysteine residues in the amino acid sequence of Renilla luciferase suggested that its secreted form could contain oxidized sulfhydryls, which might impair enzyme activity. In this work, four secreted Renilla luciferase mutants were constructed to investigate this possibility: three luciferase mutants in which a different cysteine residue was replaced by an alanine residue, and one luciferase mutant in which all three cysteine residues were replaced by alanine residues. Simian cells were transfected with the genes encoding these mutant luciferases, as well as with the original gene construct, and cell culture media were assayed for bioluminescence activity. Only media containing a mutated luciferase with a cysteine to alanine substitution at position 152 in the preprotein showed a marked increase in bioluminescence activity when compared to media containing the original secreted Renilla luciferase. This increase in light emission was due in part to enhanced stability of the mutant enzyme. This new enzyme represents a significant improvement in the sensitivity of the secreted Renilla luciferase assay for monitoring gene expression.     

In Planta Transformation of Tomato

In this study, in planta transformation of tomato (Solanum lycopersicum L.), using fruit injection and floral dip, is reported. Agrobacterium tumefaciens strain EHA 105 containing one of three constructs, i.e., pROKIIAP1GUSint (carrying the Apetala 1 [AP1] gene), pROKIILFYGUSint (carrying the LEAFY [LFY] gene), or p35SGUSint (carrying the β-glucuronidase [GUS] gene), was used for plant transformation. For fruit injection transformation, no significant effects (p > 0.05) of the construct used were observed. The highest frequency of transformation was obtained following 48-h incubation of tomato fruit with bacterial cells harboring either one of the three constructs; transformation frequencies of 17%, 19%, and 21% for AP1, LFY, and GUS gene constructs, respectively, were obtained. When fruit maturity was evaluated in fruit injection experiments, mature red fruit resulted in higher frequency of transformants than immature green fruit with 40%, 35%, and 42% for AP1, LFY, and GUS gene constructs, respectively. For floral dip transformation, a higher number of transformants was obtained when the GUS gene construct was used instead of either the AP1 or LFY gene construct, thus suggesting a possible inhibitory effect of the flowering genes used. When flowers were transformed prior to rather than following pollination, they yielded a higher transformation frequency, 12% for the LFY construct and 23% for the GUS construct (p < 0.05), although no transformant was obtained with the AP1 gene construct. All putative GUS-positive transformants were analyzed using polymerase chain reaction and confirmed for the presence of the transgene. Compared to control plants, transgenic plants carrying either the AP1 or LFY transgene flowered earlier and showed several different morphological characters.     

Metabolic engineering of ketocarotenoid formation in higher plants

Although higher plants synthesize carotenoids, they do not possess the ability to form ketocarotenoids. In order to generate higher plants capable of synthesizing combinations of ketolated and hydroxylated carotenoids the genes responsible for the carotene 4,4' oxygenase and 3,3' hydroxylase have been transformed into tomato and tobacco. The gene products were produced as a polyprotein. Subsequent cleavage of the polyprotein, targeting of the two enzymes to the plastid and enzyme activities have been shown for both gene products. Metabolite profiling has shown the formation of ketolated carotenoids from beta-carotene and its hydroxylated intermediates in tobacco and tomato leaf. In the nectary tissues of tobacco flowers a quantitative increase (10-fold) as well as compositional changes were evident, including the presence of astaxanthin, canthaxanthin and 4-ketozeaxanthin. Interestingly, in this tissue the newly formed carotenoids resided predominantly as esters. These data are discussed in terms of metabolic engineering of carotenoids and their sequestration in higher plant tissues.