Dear George, the 35S promoter indeed is not ideal to express your GFP-tagged protein. It happend to me several times that transgenic plants were expressing my 35S::xyz-GFP protein ONLY in some cells. Even though silencing has NOT been demonstrated yet to be the reason of this phenomema, many people is convinced this is the cause. Putative silencing happens more frequently when you have multiple copies of 35S present. As others already mentioned, you should test the endogenous promoter of your protein of interest as first. If it is expressed at a low level and you do not see it, then you should use other overexpressing promoters like UB10 (ubiquitin 10) or p16. I recommend ub10, its expression is 100 times lower than 35S, there is no putative silencing at all, and the expressio is very good. You can find lots of examples in the literature. One of them is: Grefen C. et al. (2010) Plant J. 64:355-365. Go for UB10!

It supress the growth, it is a constitutive promoter, it express always, generally gene specific promoter is better, one of my senior face this problem, the growth of seedling and yield reduced. Try with gene specific promoter related to which stress you need.

George, I know that light affects transcription driven by 35S promoter. So if you changed light intensity you can obtain different level of transcription. Another possibility is gene suppression, your transgene may be degradated? Have you verified mRNA level?

Although 35S is supposed to be strong and constitutive, its expression may vary slightly depending on the tissue and growth conditions. Nevertheless, I have never had situation to lose signal completely for the construct which previously showed expression.

On the other hand studying translational control I could modulate very strongly expression of reporter gene under control of 35S by modification of 5’UTR. Please find details in my pubs:

http://www.sciencedirect.com/science/article/pii/S0168945200001953

http://onlinelibrary.wiley.com/doi/10.1046/j.1365-313X.1998.00139.x/abstract

I have seen few examples of tissue specific silencing of a transgene even though they are expressed under a strong promoter. Signal may weaker at later stages however; this is very unlikely that the transgene signal got lost from all tissue at particular stage. try to see if any technical problem. You may try to locate with confocal if you have not used it yet

From a generation to the following one you can have silencing... in general, 35S in a strong and constitutive promoter. It's not the earliest promoter ever, but this shouldn't be a problem if you work on seedlings... check transgene mRNA levels.

Are you sure they are not segregating lines and you are not analyzing wrong plants. Please check more plants before thinking about gene silencing. If you have several independent lines, please check all lines to reach to any conclusion. I also agree with Marcin and Sandeep that complete loss of signal in most of the cases is not a possibility.

I think it should not alter the transgen expression, but it some time possible. I suggest u to check mRNA expression or simply shift to specific promoter.

35s won't work in very early embryonic tissue - up to heart stage. Fluorescence is certainly reduced in older plants as some others have suggested. Silencing might occur but not generally if you have a single transgene. More common for people to refer to silencing when several e.g. fluorescent protein constructs are present but I haven't even noticed that.

Dear George, the 35S promoter indeed is not ideal to express your GFP-tagged protein. It happend to me several times that transgenic plants were expressing my 35S::xyz-GFP protein ONLY in some cells. Even though silencing has NOT been demonstrated yet to be the reason of this phenomema, many people is convinced this is the cause. Putative silencing happens more frequently when you have multiple copies of 35S present. As others already mentioned, you should test the endogenous promoter of your protein of interest as first. If it is expressed at a low level and you do not see it, then you should use other overexpressing promoters like UB10 (ubiquitin 10) or p16. I recommend ub10, its expression is 100 times lower than 35S, there is no putative silencing at all, and the expressio is very good. You can find lots of examples in the literature. One of them is: Grefen C. et al. (2010) Plant J. 64:355-365. Go for UB10!

Hi. You might indeed have gene silencing through several mechanisms. Epigenetic marks can occur on your transgene and therefore silence the expression of your transgene. I am not sure but i think that the miRNA machinery might also recognize your transgene and thus silence it. So, as already recommended, check the mRNA and/or protein (western blot) levels in your plants. Good luck!

Agree with the points from Dr. Avinash Srivastava and Dr. John Runions.

In details, if your transgenic plants are not stable, there maybe segregation, in my experiences, not every T-DNA insertion can produce enough GFP protein and signal. And another thing is to check plants of different stages, also, you can check diffrent tissues. A easy way to check if there is enough GFP protein is to do western blot using raw protein from transgenic plants ( it is easy and quick way). Or if you are not familiar with that, you can try RT-PCR for your gene ( should be over-expressed) or genotyping using GFP, 35S, or kanamycin (hygromycin) primers ( only to confirm if they are transgenic plants).

In summary, western > RT-PCR> genotyping (priority sequence).

If they are positive, try good culture condition, and young healthy plants ( I guess you do culture on media?) and try different tissues.

Good luck.

I think you have to check the expression of your gene at first, either through qPCR or RT-PCR; to confirm the expression level of your insert.When you make sure of your expression level, you can check the proper construction of your GFP fusion vector; as, sometimes, the pre-designed vector can affect the expression of GFP insert.let me explain it; because I experienced something like this before; as, when we introduced our constructed insert as 35S:::Gene x:::GFP no fluorescence was obtained, while, the introduction of the construed vector as; 35S:::GFP:::Gene x gave excellent results. I have no explanation for such variation; however, my supervisor told me that it differs sometimes.

Lot of suggestions have already poured in. I do not think that I can improve over them any further.

I do not know why the GFP expression under the 35 S promotor is fading out, although co-suppression /epigene silencing is of common occurrence. It may be due to tissue specific variation. As already suggested by many , one can check mRNA expression.. Sometimes UB10 also works well instead.

Bulk up your seeds again and they'll be just fine

Hi George, I agree with Viotti. 35S is not overexpressing your protein of interest in all cells homogeniouly. The absence of GFP signal probably means silencing. This is happens quiet often depending on the insertion site. You might need to go back to the original batch of seeds two or three generations back to rescue the line. For future I might consider using UBQ10 promoter instead of 35S that allows homogenous expression in all cells although it is that strong as 35S. best of luck.

We use DsRed as reporter mostly, since it is bright and we can screen positive seeds directly, using hand held LED super bright light and a goggles with filter that limit autoflurescence! But the general idea of silencing is commun, particularly strong Pr, having an intron does help, and using fused promoters with HSP promoter does lessen silencing.

Dear George, When I grow Arabidopsis transgenic plants with 35S2 driving transgene expression I have seen difference in the transgene relative expression under in vitro and in vivo growing conditions and among plants treated with different stresses very often. I would suggest checking the expression level of the transgene. Good lack!

Dear all, thank you very much for your ideas

@John. You are right. I forgot to mention that the seeds I imbibed were stored for over 3 years. I was just confused with the expression of the transgene as their germination was perfectly OK and I thought that everything else should be OK too.

@Corrado, Anahit, Munusamy. We have also noticed mosaic patterns of expression. Unfortunately the protein we use has no homologue in Arabidopsis (it is just a marker) therefor it is not possible to clone the fusion under a native promoter. Nevertheless, we are now cloning different sets of marker proteins from Arabidopsis to overcome such problems. The ubiquitin promoter is also an attractive option

@Mustapha. What a brilliant idea. We also use DsRed and your screening approach will make things a lot easier

@Pierre. This is definitely our next step. I did not check transcript/protein levels, because I never thought to have such problems. When initially checked, transgenes were expressing beautifully (microscopy + Western)

We always see a decrease in flouresecence in the 35S line after couple of generation. For sure the silencing is an issue.

If you have many transgenic lines check more lines because GFP expression may be different in plants transformed with the same vector.

Dear all

George mention this: " that the seeds I imbibed were stored for over 3 years".

My idea is that you have change the medium and so, you change the materials' environement. For the GFP model, as physicist, I have been proposing that GFP is nothing more tha an interface photon emission. This A/B generic emitting interface can be as the one proposed within the ppt file enclosed. If you change the medium (A &/or B) or the liquid where your system is diluted, the interface emission should change or desappear.

Why I'm proposing a new discussion on the GFP model?

I do have some ideas below:

Fluorescence Resonance Energy Transfer (FRET) mechanism has been described/proposed about 60 years ago by Theodor Forster, using classical physics concepts. It is applied to explain transfer of energy from a donor molecule to a nearby acceptor molecule on photosynthetic systems (e.g.: energy harvesting on plants). FRET mechanism is a radiationless transmission of energy from a donor molecule to an acceptor molecule. According to the yet developed theory, the resonant dipole interaction between organic molecules can occurs over great than interatomic distances, without any molecular collision. The transfer of energy leads to a reduction in the donor’s fluorescence intensity and excited state lifetime, and an increase in the acceptor’s emission intensity. We describe below some drawbacks of FRET, see by a physicist:

a) The energetic configuration, used to model and to represent FRET, does not exist in physics.

b) Electrical charges do not ‘see’ the proposed energetic configuration for FRET model.

c) Electrical charges ‘see’ energetic steps (up or down), when travelling (or not) from one material to its neighbourhood.

d) Why should a molecule, after receiving energy, emit at lower energy regardless of the main peak (higher energy) it usually emits light? Density of states and statistics are not respected.

e) If the molecule with the lower bandgap energy has its actual energetic position higher than the nearby molecule, how to explain FRET without violating physics?

f) The described FRET mechanism does not separate (e-, h+). Why should Nature use a ‘step mother’ to separate (e-, h+) pairs?

g) In others words: If FRET is used to explain energy transfer from a molecule A to the nearby molecule B and this molecule B does not have the necessary physical tools to separate (e-, h+); … so what is the utility of FRET? Note that the absorption of energy (from photons or from transfer) gives to this molecule B an excitonic state but not a separated (e-, h+) pair.

h) FRET technology suffers from few drawbacks, considering unknown parameters, correction factors, intensities corrections, etc (1,2).

We propose a different model to explain energy transfer between two different organic or inorganic material’s pairs, based on the existing and actual band gap energy relative position of both involved materials. We propose that FRET is an interface solid-state-like physical mechanism. The proposed model is not based on classic physical concepts (e.g. resonance dipole interaction). Instead, it is a quantum mechanics controlled mechanism. As FRET, as described, is a radiationless energy transmission mechanism, we propose that the actual physical/optical mechanism is a radiation interface emission mechanism (3).

1- J. Szollosi et al. Cytometry 34, 159-179, 1998.

2- S. Weiss. Science 12 March, 1676-1683, 1999.

3- M. Sacilotti et al, WJNSE: http://www.scirp.org/journal/wjnse/ .

Ppt file

Hey George,

As much as it is a workhorse, the CaMV35s promoter is really rather weak. If you want big green switch to a Ubiquitin translational fusion promoter.

Dear George, I hope you might have got the solution, I would like to suggest you that one you check with blotting technique for the gene presence or lost then look for the promoter.

Hi,

growth condition of Arabidopsis mostly doesnt effect the expression of transgene unless the genetic element in the transgene has specific response to some specific environmental signal.

The most possible reason for ur GFP transgenic line could be that after some generations some transgenic line get into auto silencing phenomenon of the plant. this happens when the plant decides to auto silence the transgene. I dont have an idea how to comeover, just consult experts.