Green fluorescent protein-labeled recombinant fluobody for detecting the picloram herbicide

A green fluorescent protein-labeled fluobody was designed to develop a simple immunoassay method for detecting picloram herbicide in an environmental sample. The gfp gene was successfully inserted into the pSJF2 vector harboring the picloram-specific antibody fragment to yield pSJF2GFP. Picloram spiking in an environmental river sample could be indirectly detected by observing the fluorescence intensity value of the gfp-fluobody, exhibiting specific sensitivity to free picloram with an IC50 value of 50 ppb. Using the gfp-fluobody immunoassay avoids the enzyme-substrate reaction for calorimetric detection that is required in an enzyme-linked immunosorbent assay (ELISA).     

Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay

A solid-phase enzyme immunoassay for prostaglandin D₂ (PGD₂) was developed in which PGD₂ was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labelled and free PGD₂, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)- propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3 – 100 pg for PGD₂. The IC₅₀ value for PGD₂ in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enyzme immunoassay was applied to the measurement of PGD₂ content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD₂. PGD₂ contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay (r = 0.966) after raising its contents by intravenous administration of PGD₂. The level of PGD₂ in rat brain was extremely low but determined to be 0.11 ± 0.03 ng/g tissue (mean ± S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change.     

Stable transformation of sunflower (Helianthus annuus L.) using a non-meristematic regeneration protocol and green fluorescent protein as a vital marker

Stable transformation of sunflower was achieved using a non-meristematic hypocotyl explant regeneration protocol of public inbred HA300B. Uniformly transformed shoots were obtained after co-cultivation with Agrobacterium tumefaciens carrying a gfp (green fluorescent protein) gene containing an intron that blocks expression of gfp in Agrobacterium. Easily detectable, bright green fluorescence of transformed tissues was used to establish an optimal regeneration and transformation procedure. By Southern blot analysis, integration of the gfp and nptII genes was confirmed. Stable transformation efficiency was 0.1%. From 68 T₁ plants analyzed, 17 showed transmission of transgene DNA and 15 of them contained the intact gfp gene. Expression of gfp was detected in 10 T₁ plants carrying the intact gfp gene using a fluorimetric assay or western blot analysis. Expression of the nptII gene was confirmed in 13 T₁ plants. The transformation system enables the rapid transfer of agronomically important genes.     

Inhibition of Real-Time PCR in DNA Extracts from Aquifer Sediment

Variation in inhibition of real-time PCR was investigated with DNA extracts from 50 aquifer sediment core samples of 5 cm length collected through a 2.5 meter vertical profile across a landfill leachate plume. The inhibition was quantified using an internal control of the green fluorescent protein ( gfp ) gene, which was spiked into the real-time PCR reactions. The inhibition was investigated at two gfp gene concentrations: at 1.7 · 10 ⁷ gfp gene copies/g sediment (5.1 · 10 ⁴ gfp gene copies/PCR reaction) and at 1.7 · 10 ⁵ gfp gene copies/g sediment (5.1 · 10 ² gfp gene copies/PCR reaction). Despite the low TOC content of the sediment (average 0.4 mg C/g dw) the average real-time PCR response was partially inhibited, compared to a reference (pure water), at both high and low gfp concentrations. The relative amplification (reference = 1) was 0.85 ± 0.20 (high) and 0.66 ± 0.23 (low), showing significantly (P < 0.05) stronger inhibition at the lower target gene concentration. The inhibition of the real-time PCR did not show a systematic variation in the vertical profile related to plume position but variations were significant on a small scale of 5–15 cm depth intervals. One of the 50 samples failed to produce a signal with either concentration of the gfp internal control and three other samples inhibited real-time PCR at both high and low gfp concentration. These 4 samples, which were the samples with the highest inhibition, were the only DNA extracts with a visible brown colouration, indicating contents of humic-like substances. Elevated absorbance at 400 nm of these samples also indicated that humic-like substances were responsible for inhibition. However, other factors not associated with either absorbance or TOC may have contributed to the inhibition in less inhibited samples since the variation in real-time PCR response could not be sufficiently explained by absorbance or TOC. The results of this study suggest that an internal control is needed in real-time PCR reactions with DNA from environmental samples due to variation in inhibition to correctly quantify the number of target genes, especially at low target gene concentrations, when dilution of DNA extracts is not practical.     

Development of highly efficient genetic transformation protocols for table grape Sugraone and Crimson Seedless at low <Emphasis Type="Italic">Agrobacterium</Emphasis> density

Highly efficient genetic transformation protocols and the regeneration of transgenic plants of Sugraone and Crimson Seedless grapevines (Vitis vinifera L.) were achieved from embryogenic calli co-cultured with low Agrobacterium tumefaciens densities. The sensitivity of embryogenic cultures to kanamycin, as well as the effect of Agrobacterium strains, C58(pMP90) or EHA105, and the bacterial concentration (0.06 or 0.2 at Optical Density OD₆₀₀) on transformation efficiency were studied. Embryogenic cultures showed different kanamycin sensitivities and the total suppression of embryo differentiation at 20 and 50 mg/l kanamycin for Crimson Seedless and Sugraone, respectively. sgfp gene expression was evaluated in callus co-cultured with each bacterial strain. Although GFP transient expression was higher with A. tumefaciens EHA105 in both cultivars at the beginning of the culture, there were no significant differences 28 days post-inoculation. However, the concentration of Agrobacterium did affected transformation efficiency: 0.06 OD₆₀₀ being more effective for the transformation of Crimson Seedless and 0.2 OD₆₀₀ for Sugraone. By following the optimised procedure, 21 and 26 independent transgenic plants were generated from Sugraone and Crimson Seedless respectively, three to five months post-infection. PCR analyses were carried out to verify the integration of the sgfp and nptII genes into grapevine genome and the stable integration of the sgfp gene was confirmed by Southern blot.     

Optimization of nutrient levels in the medium increases the efficiency of callus induction and plant regeneration in recalcitrant indian barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) <Emphasis Type="Italic">in vitro</Emphasis>

Summary The present study reports that a revised nutrient concentration in the basal medium improved shoot bud induction and subsequent plant regeneration in barley (Hordeum vulgare L. var. BL-2). Cultures were raised from immature embryos on MSB₅ medium supplemented with picloram. Concentrations of five nutrients were varied. The effect of these nutrients was investigated on (1) induction, (2) induction and subculture, and (3) induction, subculture and regeneration stages. The basal MSB₅ medium was not optimal for each phase of barley culture. Decreased ammonium nitrate, increased potassium dihydrogen phosphate, sodium molybdate, cobalt chloride, and addition of glycine enhanced shoot bud induction and plant regeneration. The different media that were optimal for immature embryo culture were: MSB₅ medium supplemented with 20.70 μM picloram, 10.30 mM NH₄NO₃, 6.25 mM KH₂PO₄, 2.06 μM Na₂MoO₄, 0.55 μM CoCl₂, and 26.64 μM glycine (for induction); MSB₅ medium supplemented with 12.47 μM picloram, 10.30 mM NH₄NO₃, and 0.55 μM CoCl₂ (for subculture); and MSB₅ medium supplemented with 0.2 μM picloram and 10.3 mM NH₄NO₃ (for regeneration). Primary cultures required 6wk (without transfer) for morphogenic callus formation. Callus required 4wk of subculture and another 4wk on regeneration medium for optimal plant regeneration. The revised medium could also promote regeneration of the recalcitrant barley genotype RD-2552. Histological analysis showed that the major pathway of differentiation was through shoot bud formation.     

Effect of several herbicides on bacterial populations and activity and the persistence of these herbicides in soil

Summary Samples of a sandy loam soil were supplied with normal, 10-fold and 100-fold rates of ioxynil, dalapon, mecoprop, dichlorprop, MCPA + dichlorprop, picloram, and amitrole-T and incubated at 29°C at 65 per cent of the waterholding capacity. Treated soil samples were compared with untreated samples. Samples supplied with (NH₄)₂SO₄ and herbicides were used to investigate the effect of the herbicides on the rate of nitrification and the production of nitrite. In several cases higher numbers of bacteria were found for a longer or shorter period in soil treated with herbicides. There was some evidence that certain groups of bacteria had adapted to ioxynil in a soil sample supplied with the 100-fold rate of this chemical. After 2 or 4 weeks lower numbers ofAzotobacter chroococcum were found at the normal rate of ioxynil, dalapon, mecoprop, and dichlorprop. At the 100-fold rate of application the numbers of Azotobacter were unfavourably affected by all herbicides. The production of mineral nitrogen was hardly affected by the normal and 10-fold rates of application. In the first week the rate of nitrification was slightly depressed in soil samples treated with the normal rates of dichlorprop and amitrole-T and with the 10-fold rates of dalapon, mecoprop, and MCPA + dichlorprop. Strong inhibition of the nitrification for at least 7 weeks was shown by the 10-fold rate of amitrole-T. At the 100-fold rate all herbicides, with the exception of picloram, depressed the rate of nitrification for a longer or shorter period. During the second week a very small increase of nitrite was found in the samples treated with the normal and 10-fold rates of dalapon, mecoprop, dichlorprop, and amitrole-T. A small increase of nitrite was noted for 26 weeks in samples treated with the 100-fold rates of amitrole-T. A highly significant depression of CO₂ evolution was found in the first week in samples treated with the normal rates of ioxynil, dalapon, mecroprop, dichlorprop, and amitrole-T, also in samples treated with the 10-fold rates of dalapon, mecoprop, dichlorprop, MCPA + dichlorprop, picloram, and amitrole-T. A highly significant depression of CO₂ production was found after 8 weeks in all the samples treated with herbicides at the 100-fold rate with the exception of the sample treated with picloram. The decomposition of the herbicides was studied in soil samples treated with the 100-fold rates of herbicides. Only traces of dalapon and mecoprop were found after 9 months, but 7.2% ioxynil, 29.8% dichlorprop, 39% (MCPA + dichlorprop), 52.1% picloram and 52.2% amitrole-T were still present in active form.     

Fluorescent reference strains of bacteria by chromosomal integration of a modified green fluorescent protein gene

Fluorescent reference strains of bacteria carrying a stable chromosomally integrated single copy of the gfp gene have been developed. A modified version of the gfp gene has been generated by mutagenesis and expressed under the control of the bacteriophage lambda promoter P_L. A cassette comprising bacteriophage Mu transposon arms flanking the modified gfp gene and regulatory regions was irreversibly integrated as an in-vitro-assembled transposition complex into the genomes of Escherichia coli and Salmonella spp. The modified green fluorescent protein (GFP) protein retained the fluorescence excitation and emission wavelengths of wild-type GFP. However, it fluoresced more brightly in E. coli and Salmonella compared to wild-type GFP, presumably due to improved protein maturation. Fluorescent E. coli and Salmonella strains carrying the gfp gene cassette were easily differentiated from their respective non-fluorescent parental strains on various growth media by visualization under UV light. The bacterial strains produced by this method remained viable and stably fluorescent when incorporated into a matrix for delivery of exact numbers of viable bacterial cells for use as quality control agents in microbiological procedures.     

Visualizing the infection process of Xanthomonas campestris in cabbage using green fluorescent protein

The plant pathogen, Xanthomonas campestris NRRL B-1459 was chromosomally tagged with gfp, and the transformant, which was subjected to Southern hybridization showed the presence of gfp in the chromosome. The virulence-related gene of the transformant was not affected by the insertion of gfp. After inoculation into cabbage plants, the infection process was visually studied in planta. Using a fluorescence microscope, the migration and distribution of gfp-labelled bacteria was visualized in real time. As the gfp-labelled cells were easily visualized from the beginning of infection, we observed a time delay of 2 days between distribution of the Xanthomonas cells in cabbage plant and the appearance of visible necrosis.     

Expression of the green fluorescent protein carried by Autographa Californica multiple nucleopolyhedrovirus in insect cell lines

Summary A recombinant AcMNPV containing the green fluorescent protein (gfp) gene under the polyhedrin promoter (polh) was used to investigate the expression of the gfp gene as well as the production of recombinant extracellular virus in 14 continuous insect cell lines, including Heliothis virescens (BCIRL-HV-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), Spodoptera exigua (BCIRL/AMCY-Se-E1 and BCIRL/AMCY-Se-E5), Bombyx mori (BMN), Sf9 (a clone of IPLB-SF21), and five cell line clones of BCIRL-HV-AM1. The susceptibility of the cell lines to the recombinant virus (AcMNPV.GFP) was ascertained by calculating the mean percentage number of green light-emitting cells as well as by TCID₅₀ titration of extracellular virus with fluorescence as a sign of infection. Of the 14 cell lines tested, all were permissive with varying degrees to Ac-MNPV.GFP, except BCIRL-HV-AMCL2 and BCIRL-HZ-AM1, both grown in serum-containing medium, and BMN, grown in serum-free medium, which were nonpermissive to the virus. Except for BCIRL/AMCY-Se-E1, IPLB-SF21, and four of the five BCIRL-HV-AM1 clones, all the other cell lines (BCIRL-HV-AM1, BCIRL-AG-AM1, TN-CL1, Se-E5, and Sf9) expressed detectable levels of GFP by 48 h postinoculation. The BCIRL/AMCY-Se-E1 and IPLB-SF21 cells, grown in serum-free medium (Ex-Cell 401), expressed detectable levels of GFP at 72 h postinoculation. By contrast, in BCIRL/AMCY-Se-E1 in serum-containing medium (Ex-Cell 401+10% FBS [fetal bovine serum]), GFP was detected at 48 h postinoculation. Furthermore, TN-CL1 cells produced the largest mean percentage number of fluorescent (76.6%) cells in both serum-containing and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. The mean extracellular virus (ECV) production at 120 h postinoculation was highest in BCIRL/AMCY-Se-E5 cells grown in Ex-Cell 401+10% FBS (37.8×10⁶ TCID₅₀/ml) followed by BCIRL-HV-AM1 in TC199-MK (33.4×10⁶ TCID₅₀/ml). Only the BCIRL-HV-AMCL3 clone produced any substantial level of ECV at 120 h postinoculation (16.9×10⁶ TCID₅₀/ml). However, there was no significant correlation between ECV production and the mean percentage number of fluorescent cells. This study provides further information on the susceptibility of 14 insect cell lines to a recombinant AcMNPV containing the green fluorescent protein gene. This information might avail researchers with information to facilitate decisions as to what other cell lines are available for in vitro studies of the gfp gene.     

Detection of 3-phenoxybenzoic acid in river water with a colloidal gold-based lateral flow immunoassay

3-Phenoxybenzoic acid (3-PBA) is a general metabolite of synthetic pyrethroids. It could be used as a generic biomarker for multiple pyrethroids exposure for human or pyrethroid residues in the environment. In this study, monoclonal antibodies (mAbs) against 3-PBA were developed by using PBA–bovine serum albumin (BSA) as an immunogen. In the competitive enzyme-linked immunosorbent assay (ELISA) format, the I₅₀ and I₁₀ values of purified mAbs were 0.63 and 0.13 μg/ml, respectively, with a dynamic range between 0.19 and 2.04 μg/ml. Then, the colloidal gold (CG)-based lateral flow immunoassay was established based on the mAbs. The working concentration of coating antigen and CG-labeled antibodies and the blocking effects were investigated to get optimal assay performance. The cutoff value for the assay was 1 μg/ml 3-PBA, and the detection time was within 10 min. A total of 40 river water samples were spiked with 3-PBA at different levels and determined by the lateral flow immunoassay without any sample pretreatments. The negative false rate was 2.5%, and no positive false results were observed at these levels. This lateral flow immunoassay has the potential to be an on-site screening method for monitoring 3-PBA or pyrethroid residues in environmental samples.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation and regeneration of transgenic plants using leaf segments as explants in Valencia sweet orange

In this study, attempts were made to develop a protocol for regeneration of transgenic plants via Agrobacterium tumefaciens-mediated transformation of leaf segments from ‘Valencia’ sweet orange (Citrus sinensis L. Osbeck) using gfp (green fluorescence protein) as a vital marker. Sensitivity of the leaf segments regeneration to kanamycin was evaluated, which showed that 50 mg l⁻¹ was the best among the tested concentrations. In addition, factors affecting the frequency of transient gfp expression were optimized, including leaf age, Agrobacterium concentration, infection time, and co-cultivation period. Adventitious shoots regenerated on medium containing Murashige and Tucker basal medium plus 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.5 mg l⁻¹ 6-benzyladenine (BA) and 0.5 mg l⁻¹ kinetin (KT). The leaf segments from 3-month-old in vitro seedlings, Agrobacterium concentration at OD₆₀₀ of 0.6, 10-min immersion, and co-cultivation for 3 days yielded the highest frequency of transient gfp expression, shoots regeneration response and transformation efficiency. By applying these optimized parameters we recovered independent transformed plants at the transformation efficiency of 23.33% on selection medium (MT salts augmented with 0.5 mg l⁻¹ BA, 0.5 mg l⁻¹ KT, 0.1 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 250 mg l⁻¹ cefotaxime). Expression of gfp in the leaf segments and regenerated shoots was confirmed using fluorescence microscope. Polymerase chain reaction (PCR) analysis using gfp and nptII gene-specific primers further confirmed the integration of the transgene in the independent transgenic plants. The transformation methodology described here may pave the way for generating transgenic plants using leaf segments as explants.     

Monitoring ofgfp-taggedMoraxella sp. under starvation condition

A PNP(p-nitrophenol)-degradingMoraxella sp. was genetically marked bygfp gene for monitoring. Stable chromosomal integration of the introducedgfp gene was confirmed by examining the transformants under epifluorescent microscope. The survival ofgfp-taggedMoraxella sp. cells during long-term storage under starvation condition was examined by viable cell counting and direct fluorescence microscopic counting. The number of green fluorescent cells obtained by direct microscopic counting was approximately 10 times greater than viable cell counts by plating. The number of cells from both counting methods was higher at lower temperature (4°C), although the drop of cell number after 8 weeks of starvation was comparable (approximately 100 fold drop from initial counts). Results obtained by two different methods correlated well with each other indicating that thegfp markedMoraxella sp. can be directly monitored following environmental release using epifluorescence microscopy.     

dsRNA-induced gene silencing in Moniliophthora perniciosa, the causal agent of witches’ broom disease of cacao

The genome sequence of the hemibiotrophic fungus Moniliophthora perniciosa revealed genes possibly participating in the RNAi machinery. Therefore, studies were performed in order to investigate the efficiency of gene silencing by dsRNA. We showed that the reporter gfp gene stably introduced into the fungus genome can be silenced by transfection of in vitro synthesized gfpdsRNA. In addition, successful dsRNA-induced silencing of endogenous genes coding for hydrophobins and a peroxiredoxin were also achieved. All genes showed a silencing efficiency ranging from 18% to 98% when compared to controls even 28 d after dsRNA treatment, suggesting systemic silencing. Reduction of GFP fluorescence, peroxidase activity levels and survival responses to H₂O₂ were consistent with the reduction of GFP and peroxidase mRNA levels, respectively. dsRNA transformation of M. perniciosa is shown here to efficiently promote genetic knockdown and can thus be used to assess gene function in this pathogen.     

Enhanced chemiluminescent sandwich enzyme immunoassay for hen egg lysozyme

A sensitive chemiluminescent sandwich-type enzyme immunoassay for hen egg lysozyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conjugate and the H₂O₂/luminol-enhanced chemiluminescence detection reagent. The chemiluminescent signal was detected using either a microplate luminometer, or photographic film in a camera luminometer. The detection limit for lysozyme was 0.3 ng/mL, and this was three times lower than that obtained using a colorimetric method with H₂O₂ and o-phenylendiamine as substrates. Recovery of the assay was 97–112% and the relative standard deviation ranged from 3.6% to 10.3%. The immunoassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.     

Construction of a Tightly Regulated Plasmid Vector for Streptococcus pneumoniae: Controlled Expression of the Green Fluorescent Protein

We have constructed a regulated plasmid vector for Streptococcus pneumoniae, based on the streptococcal broad-host-range replicon pLS1. As a reporter gene, we subcloned the gfp gene from Aequorea victoria, encoding the green fluorescent protein. This gene was placed under the control of the inducible P_M promoter of the S. pneumoniae malMP operon which, in turn, is regulated by the product of the pneumococcal malR gene. Binding of MalR protein to the P_M promoter is inactivated by growing the cells in maltose-containing media. Highly regulated gene expression was achieved by cloning in the same plasmid the P_M-gfp cassette and the malR gene, thus providing the MalR regulator in cis. Pneumococcal cells harboring this vector gave a linear response of GFP synthesis in a maltose-dependent mode without detectable background levels in the absence of the inducer.     

Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfp uv ) from Escherichia coli

Background  

Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside), express the green fluorescent protein (gfp _uv ) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP) method were compared to the direct application of TPP to the same culture of E. coli on releasing gfp _uv from the over-expressing cells.     

The actinobacterium <Emphasis Type="Italic">Microbacterium</Emphasis> sp. 16SH accepts pBBR1-based pPROBE vectors,forms biofilms,invades roots,and fixes N<Subscript>2</Subscript> associated with micropropagated sugarcane plants

Members of the genus Microbacterium lineage of Gram-positive actinobacteria are increasingly being reported to display significant traits associated with environmental biotechnology and bioengineering. 16SH is a nitrogen-fixing bacterial strain isolated from a surface-sterilized stem of sugarcane grown in Guangxi, China. Analysis of 16S rRNA gene sequences revealed that 16SH belonged to the genus Microbacterium. pPROBE-pTet^r plasmids were constructed by cloning the promoter region of the Tet ^r gene into the promoterless pPROBE-AT, -OT, and -TT vectors derived from the pBBR1 plasmid that has a broad host range of Gram-negative bacteria and sequence similarities to plasmids from Gram-positive bacteria. The pPROBE-pTet^r plasmids expressed the gfp reporter gene and were stably maintained in 16SH cells without antibiotic selection in free-living state and in planta. Confocal microscopy on intact roots of micropropagated sugarcane plantlets showed that gfp-tagged 16SH cells formed biofilms on root maturation and elongation zones but not on root meristem zones and root caps, and colonized in intercellular spaces of root cortices. Inoculation of 16SH significantly increased biomass and nitrogen content of micropropagated sugarcane seedlings grown with a nitrogen fertilization of 6.3 mg N/kg soil. ¹⁵ N isotope dilution assays demonstrated that biological nitrogen fixation contributed to this plant growth promotion. This study for the first time demonstrated that the pBBR1-based pPROBE plasmids provided an efficient genetic transfer system for a Gram-positive Microbacterium strain, and that a nitrogen-fixing Microbacterium endophyte colonized in intact host plants and fixed N₂ associated with the host plants.     

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing,cell death and T-DNA loss

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells fromAgrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.     

Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system

To test the Cre/loxP recombination system in zebrafish, a stable transgenic zebrafish line was developed by using a floxed (loxP flanked) gfp(green fluorescent protein) gene construct under the muscle-specific mylz2 promoter. Like our previous non-floxed gfp transgenic line under the same promoter, the new transgenic line expresses GFP reporter faithfully in fast skeletal muscles to the same intensity. To demonstrate the excision of floxed gfp transgene, in vitro synthesized Cre RNA was injected into embryos of floxed gfp transgenic zebrafish and we found a dramatic reduction of GFP expression. To confirm the excision, PCR was performed and a DNA fragment of correct size was amplified as predicted from the Cre/loxP mediated excision. Finally, we cloned the fragment and sequence information confirmed that the excision occurred at the precise site as predicted. Our experiments demonstrated that the Cre/loxP system can function efficiently and accurately in the zebrafish system.