ngin - Norfolk Genetic Information Network

23 September 2002


source: "roger mainwood" <

Earlier this year I had the folowing reply from Dr Jane Cockram at the Food Standards Agency.

It concerned a FSA financed study carried out at the John Innes Centre. She wrote............

Mr R Mainwood
roger.mainwood@virgin .net
17 June 2002 Reference: NFU76
Dear Mr Mainwood
Research Project G01009: Impact of transformation methods, construct and gene cassette architecture on the stability and expression of transgenes.
Thank you for your emails of 4 and 20 May 2002 regarding the results of the above research project.
You asked specifically about the role of introns and how the process of genetic modification effects these sequences. As the project in question has only carried out experiments in barley and wheat then the results observed are relevant to these plant species only.
On a more general notes you asked about what we now know about the effect of genetically modifying plants and in particular the forced introduction of introns. All the research conducted to date on this subject has provided background information to the process of genetic modification. Before being approved for food use or deliberate release into the environment all GM crops undergo a rigorous safety assessment.  This assessment includes an examination of the sequences inserted into the plant and their effect on the expression and stability of the transgene. Each assessment is therefore carried out individually on a case by case basis.
  Yours sincerely
  Dr Jane A Cockram
  Novel Foods Division

........well I thought this wasn't very satisfactory, and so I asked Dr Michael Antoniou (Senior Lecturer in Molecular Genetics, GKT Medical School, Guy's Hospital, London ) for his opinion. This is his illuminating reply.........


From Dr. Michael Antoniou
Date: 17 September 2002 10:12
Subject: Fw: John Innes report
Dear Roger

It has been many months now since you sent me your original enquiry. I'm very sorry for taking so long to respond; I'm afraid that it simply got buried in a pile and got "lost"! Anyway, I hope this reply is not too late for whatever purpose you had in mind to use the information in this John Innes report.

I found this a very interesting report to read and wouldn't mind a full copy at some point; I'll contact the John Innes Centre for this or it may even be on their web site.

So what does this study tell us?

Firstly, the reply you got from Dr Jane A Cockram at the FSA is totally non-descript and actually shows a lack of understanding of the basic principles of molecular biology on which this type of GM plant technology is based. She says "As the project in question has only carried out experiments in barley and wheat then the results observed are relevant to these plant species only." This is nonsense as the project was addressing general principles of the stability of transgene function in GM plants and particularly the role of introns in this process; i.e. if introns could improve the stability of transgene expression. So, this work has major implications for GM plant production in general. The rest of her message is just civil servant government gobbledygook with no technical meaning as we know!

"clean-gene system"

What they mean by "clean-gene system" is that using their newly developed Agrobacterium mediated gene transfer system in wheat and particularly in barley, they will be able to generate transgenic varieties of these two plants that will NOT contain antibiotic resistance marker genes. The plants will contain only the transgene of interest for the intended change which in their minds is now a more "clean" product! However, the plants will still be "dirty" in all other respects. The presence of the intended foreign transgene alone can still cause disruptions to host gene function and cause various problems from reduced crop performance to the generation of novel toxins and allergies, etc. Monsanto's famous/infamous Roundup Ready Soya beans are such an example of a "clean" GM plant as it does not contain an antibiotic resistance marker gene and only the Roundup herbicide resistance gene. Despite this it is now well documented that various varieties of RR Soya have suffered all kinds of agronomic problems including, most importantly for the farmer, an average reduced yield of 10%, at least 5% of which can be attributed to disruption of plant biology by the random insertion and presence of the foreign gene (see the NLP Wessex web site for details).

Variable transgene function between generations

For me the most interesting and important aspect of this study is that it demonstrates very clearly in a controlled manner the unpredictability of GM technology highlighted by extremely variable transgene function from generation to generation. The level of transgene function (expression to give rise to the protein product the gene codes for), varied from generation to generation for ALL the transgenic wheat and barley plants they studied. This shows that depending on where the transgene has spliced into the plant DNA, the efficiency with which it is switched-on and to work varies not only in the initial GM parent but also in the subsequent generations of plants produced (after flowering/seeding etc) from this parent. So much for GM plant technology being precise! This very nicely highlights all we have been saying all along that GM is imprecise and unpredictable.

Unfortunately, the parts of the report you sent me does not say whether the authors of the study looked into the molecular mechanisms behind this variability in transgene function between generations. There are various mechanisms that could give rise to this including disruption of the transgene by recombination. But no molecular genetic analysis of the plants was carried, from what it says, to find out whether recombination or other processes were taking place. The inclusion of introns in their transgenes certainly did not improve stability of function in a reproducible manner ("By adding specific additional sequences to the constructs (additional introns) it was possible to either increase or decrease expression."). I normally include introns in the mammalian transgenes that I work with as this generally helps in efficiency of expression and we do not observe instability due to enhanced recombination due to the presence of introns. But then I'm working in completely different types of organisms!! My personal feeling is that the presence of introns is probably not enhancing recombination in these plants and that the silencing of transgene function/expression between generations is by other mechanisms.

So, I think in summary the best way we can use this interesting piece of work as it stands, is to use it as another illustration of just how imprecise and unpredictable plant GM technology can be.

I hope this helps and apologies again for the long delay in my response.

With very best wishes


For reference:-

  The report (Project GO 1009) is titled:  "The impact of transformation methods, construct and gene casette architecture on the stability and expression of transgenes."
  The study, carried out at the John Innes Centre, was originally funded by MAFF (now DEFRA) as part of a series of projects addressing the stability and expression of genes introduced into genetically modified (GM) plants. The aim of the project was to look at different methods of transforming plants and the design of the construct used to introduce a gene into the plant and how these parameters influence the stability and expression of the introduced gene. In the project an introduced marker gene (which produced a fluorescent protein) was studied in GM barley and wheat.
  I have now obtained a copy of the report (£9 from the Food Standard Agency library) and and here is the non-technical executive summary that it contains.........
  Impact of transformation methods, construct and gene cassette architecture on the stability and expression of transgenes.
  Dept of Crop Genetics
  John Innes Centre
  Start Date: 01/10/1998
  End Date: 31/12/2001
  Final Technical Report for project G01009 -  MAFF/DEFRA funded
  Staff who have contributed to the project:
  Prof John Snape
  Dr David Lonsdale
  Dr Wendy Harwood
  Non-technical Executive Summary
  Rationale and Objectives:
  This project was set up to firstly look in detail at GM wheat and barley lines to determine how the expression of a particular introduced marker gene (the firefly luciferase gene) changed over at least four generations in GM plants.
  Secondly the project aimed to study the effect of changing the introduced DNA in ways expected to have an effect on stability and expression of the introduced gene. Some of the changes made were expected to have an effect on the stability of expression of the introduced gene from one generation to the next (for example adding matrix attachment regions and additional introns). The effects of these modifications was then evaluated.
  As part of this project a range of different "constructs", that is DNA in a form suitable for inserting into plants to obtain GM plants, were made. In addition, improved methodology for the production of GM barley plants was developed. This was done by developing a reproducible Agrobacterium-mediated transformation system for barley as an alternative to the direct gene delivery system normally used. The Agrobacterium-mediated transformation system has the potential toallow the production of GM plants that do not contain marker genes.
  The aim of this project was to provide a better understanding of the factors that influence the expression of transgenes (the introduced genes in GM plants) and of how transgene expression and stability may vary over several generations in GM wheat and barley.
  The first part of this project involved producing a number of GM wheat and barley lines. These were grown in containment glasshouses together with a number of lines that had been previously produced, for four or five generations. At each generation, leaf samples were collected and a range of molecular and biochemical analysis methods were used to determine whether the introduced genes had been rearranged in any way and whether they were expressed in the same way as in the previous generation.
  Outcome/Key results Obtained:
  The project has provided reproducible methods for the production of GM barley using two different protocols and GM wheat using one protocol. It has also provided a range of constructs and a range of transgenic lines of wheat and barley that have been characterised and analysed over a number of generations.
  Key findings from the analysis included the observation that by selecting the highest expressing plant at each generation and analyzing the progeny from this plant, large differences in expression of the transgene were often observed from one generation to the next. Transgene expression was seen to increase or decrease between generations. In general, very high levels of expression seen in a particular generation were not maintatined in the subsequent generation. Similar patterns of changes in expression levels were seen in both wheat and barley containing the same or different constructs. It was found that in general, expression levels were much higher in barley than in wheat when the same methodology and the same constructs were used. Changing the orientation of the different components of the construct also had an effect on expression levels.
  By adding specific additional sequences to the constructs (additional introns) it was possible to either increase or decrease expression . It was also possible to affect the stability of expression over generations and the number of copies of the transgene present in the plants.
  Importance of the results:
  The results suggest that variation in transgene expression between generations in wheat and barley is likely in GM lines expressing high levels of the luciferase transgene. Such high expressing lines exhibited the largest variation in expression whereas low expressing lines showed less variation. The approach of selecting the highest expressor in each generation did not, in most cases, lead to stable expression over four or five generations and is therefore not the method of choice for identifying stable lines.
  In this project, the first steps have been taken towards controlling transgene expression levels by modifying the transform construct. In addition the development of an Agrobacterium-mediated transformation system for barley paves the way for the development of GM plants containing only the gene of interest and no marker genes, thus removing the concerns about the presence of antibiotic and other marker genes.
  You might want to read a bit more about what that last paragraph had to here is a more detailed summary from the main body of the report about what they are calling the "clean-gene system".
  " proved to be very difficult to repeat the transformation of barley using Agrobacterium. However we have recently developed a reproducible barley transformation system using Agrobacterium and have now produced large numbers of transgenic plants containing a range of vectors including pDM805, pAL135 and pVec8Gus. the first two vectors contain the bar gene as a selectable marker while pVec8Gus contains a hygromycin resistence gene. Using hygromycin for selection of transgenic tissue has proved very successful and provides a clearer selection system than the bar gene. Transformation frequencies are between 2 and 4% considering only the successful experiments. However now the methodology is working well few experiments fail to give transgenic plants. We are currently working to transfer this Agrobacterium-mediated transformation technology to wheat. The reproducible Agrobacterium-mediated barley transformation system will now allow us to develop a clean-gene transformation system for barley although this has not been achieved as part of this project. The clean-gene system relies on placing the selectable marker gene on one T-DNA and the gene of interest on a seperate T-DNA and then looking for segregation of the marker gene away from the gene of interest in the progeny."

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