Assessments are based on assumed lack of risk and lack of evidence. In the case of the CaMV 35S promoter any question of risk was immediately discounted as this promoter was regarded to be plant specific and not active in other organisms such as bacteria, fungi or human cells. This assumption is wrong see below , putting the whole current procedure of risk assessment and accuracy of company safety data in question.
Failure to recognise or to ignore its capacity to be universally active in almost any organism is irresponsible and careless and shows a serious lack of scientific rigour and commitment to safety. Consequently, in the unlikely event of gene transfer from the transgenic maize to intestinal bacteria, expression of the pat gene would not occur.
This is because it is linked to the cauliflower mosaic virus promoter that expresses genes in plants - not bacteria. Already in it had been established that the CaMV 35S promoter is not only active in plants but also in the gut bacterium E. It has more recently been shown that the promoter is active in gut pathogens i. It is vital to include such knowledge and findings if a risk assessment is to be dependable, trustworthy and scientific.
Abstract Positive selection of transgenic plants is essential during plant transformation. Publication types Research Support, Non-U. Substances Genetic Markers. Five fish from each of the treatment groups receiving pGLS or PBS were killed 7, 30, 70, and days after i.
In brief, pooled samples were pre-processed for luciferase activities, the relative emitting light chemiluminescence units RLU were normalized to the protein concentrations using measurements from Bio-Rad RC DC assay Bio-Rad Lab.
Five fish from each of the treatment receiving pGLS were killed 7, 30, 70, and days after i. At day 7, 30, 70, and the liver, kidney, spleen, heart, gills, muscle, anterior and posterior intestine and tissue surrounding the injection site were harvested for Southern blot analysis.
Total DNA was extracted from tissue samples using the E. Primers were designed using the Primer Express software version 2. In this first experiment, the fish were killed at day 7, since previous studies have shown a high degree of reporter gene expression at this time-point using several different other promoters in different animal models 15 , 16 , 17 , No luciferase activity was found in samples from the other tissues and organs sampled or in the control fish injected with PBS.
Each bar represents the mean luciferase activity of five fish per treatment group and error bars represent S. The measured luciferase activity was normalised for total protein content in muscle. P-values are inserted. The background luciferase activity in muscle controls injected with PBS was 5.
The luciferase activities from muscle samples obtained from fish injected wit pGL3-Basic, pGL3-SV40 and pGLS were approximately 22, 43 and times higher compared to the average level detected in fish injected with the PBS, respectively. Luciferase activity was not detected anywhere else than in muscle tissue injection site at the first sampling time-point at day 7 post-injection.
Consequently, examination for long-term luciferase expression was subsequently performed on the muscle tissues only. Luciferase activity was detected at the injection site up to days post-injection of pGLS Fig. Luminescence quantification of luciferase activity in Atlantic salmon muscle tissue 7, 30, 70, and days after intramuscular injection of pGLS. PBS injected fish served as control. Each bar represents the mean luciferase activity of five fish and T-bars represent the S.
The measured luciferase activity was normalised for total protein mg. Subsequent Southern blot analysis suggested the presence of two extra-chromosomally located different topoforms of pGLS in the liver, kidney, spleen, heart, gills, muscle, anterior- and posterior intestine and at the site of injection 7 days post-injection Fig.
These topoforms were presumably open circular OC approx. The presence of pGLS in the injection site at 14, 70, and days post-injection was also determined by Southern blot analysis. Linear pGLS was present in the muscle tissue at the injection site 14, 70, and days after injection —evaluated by Southern blot of samples priory digested with Pst I data not shown.
Furthermore, total DNA collected at day and from the site of injection was digested with the restriction enzyme Pvu II. Southern blot analysis suggested extra-chromosomally open circular, linear and supercoiled topoforms of pGLS at day Fig.
A sample retrieved from the stock plasmid preparation pGLS contained mostly open circular and supercoiled topoforms and minor amount of linearized topoform Fig. However, only the open circular and supercoiled topoforms were detected at day Fig. Samples obtained from control fish contained no pDNA. Southern blot showed extra-chromosomally open circular and supercoiled topoforms of pGLS in different tissues on day 7 days in the muscle of Atlantic salmon. DNA from samples of five fish from each treatment groups were mixed to yield samples for the Southern blot experiment.
Samples shown were obtained and days after injection. The measured enzymatic activity of plasmid-encoded luciferase at the injection site seven days after injection cf. We were then interested to study the long-term expression of luciferase enzyme at the injection site using this construct containing the 35S CaMV promoter. Interestingly, expression of luciferase activity was detected in the muscle tissue at the injection site even days after injection.
This is comparable with previous findings where luciferase under the control of a CMV promoter also promoted expression of luciferase for days 19 and also comparable to observation made in the glass catfish We cannot offer any explanation why the promoter- and enhancer-less pGL3 Basic induced expression of luciferase at the injection site 7 days after plasmid injection.
We have also found similar result, in a control experiment, where the pGL3-Basic was able to induce a certain luciferase activity; 0. This support the finding that pGL3-Basic may induce luciferase expression at the injection site — albeit at a low level. Previous to this study we, however, found that the measured luciferase by luminometer activity was highly dependent on the inherent tissue colour.
As a consequence, we extrapolated the luciferase activity from a standard curve made on control tissue homogenates with added recombinant luciferase. The quenching by tissue colours was higher in blood-rich tissues and tissues with strong colour, while it was close to zero in muscle tissue that did not contain much colour. This extrapolation would add a small uncertainty to the data set, but the interpretation of the results is not changed. The next research questions were whether this expression of luciferase activity was related to presence of intact plasmid DNA at the injection site.
Seven days after injection, both open circular and supercoiled plasmid DNA pGLS were found in all tissues and organs analysed. The stock solution of pGLS also contained both supercoiled and open circular conformations result not shown. The tissue processing and DNA isolation steps prior to conformational analysis have in previous study not been found to cause any strand breaks in the DNA Tonheim et al.
Although plasmid DNA was present in many different tissues at day 7, no luciferase activity was detected except at the injection site. These results are supported by the findings shown by Tonheim et al. This finding is in contrast with previous study that described luciferase expression, under CMV and activity in distally localised tissues after i. This study has confirmed for the first time in any vertebrate that a S35 CaMV promoter is able to drive expression of a transgene and that the duration of transgene production is at least 1.
How to cite this article : Seternes, T. Odell, J. Nature , —, doi: Fromm, M. Expression of genes transferred into monocot and dicot plant cells by electroporation. Assaad, F. Cauliflower mosaic virus P35S promoter activity in Escherichia coli.
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