Madhurima, thanks for clarifying that you are working with plants. You may know best, if the coding sequence of your vector insert should be OK; for example, if it came from an good and proven source, or, if there is a chance that there could be something wrong with the coding sequence: did you clone it yourself, for example, just by PCR? Then there is a chance that the subcloned sequence was wrong and not confirmed by sequencing and contains a stop codon or other mutations. But lets assume you have used a confirmed vector and sequence and the protein really induces some negative feedback, as you assume, I then could think of "in vivo footprint assays" to check if the promoter of your transgene is blocked by some proteins (blocking the known transcription factor binding sequences). This procedure would only be useful, if the RNA level is less than expected; so, I would check with a Northern blot the level of RNA of your transgene - perhaps, compared to any other cell, were the transgene worked, or compared to any other RNA within the cell. I guess, you expect tons of transgene RNA - and, for a toxin that could be a problem. Just a suggestion to think about a different (weaker) promoter for the transgene? But I guess it won't be easy, although very interesting, to find out what is really happening. I hope others have more ideas.

What vector system did you use for the expression of the protein? Did you check if the gene is in the correct reading frame after insertion into vector? If the gene is not in the correct frame, it may result in expression of truncated protein or no expression. You can run an SDS-PAGE gel to visualize the expression/over-expression of the protein.

1) There could be a point mutation in the transgene - did you sequence it?

2) RT-PCR is a good indication of your expected RNA, but does not proof that it is highly abundant (as usually depending on the promoter used). But the promoter could somehow be less active.

3) Since you use some toxin, it could harm those cells expressing (enough) protein - so you may select for the others.

4) PCR could be false-positive. A southern would proof the integration of the transgene into the genome (although it may not really help to find the cause of the translational problem).

5) Did you check just one line of transgenig (animals, cells)? Do they all have this problem?

Robert, I am working with toxic proteins and trying to develop transgenic plants with them. I am checking for my protein expression as well as transcripts in the T1 progeny lines (I am checking a number of plants). I would now like to see if my toxin is being selectively silenced? I believe that is the case and therefore there is no protein expression even though they are PCR positive. So how do I go about analyzing them now?

Sequencing is a good way...is there any other method?

If DNA sequence is correct, and there is ARN, maybe the expression level is not enough to observe a band in a SDS-PAGE, try western blot with a specific antibody.

Some proteins don't reach high levels, especially if they are toxic in some way, or could be partially digested obtained bands with a MW different from the one you expect.

Madhurima, thanks for clarifying that you are working with plants. You may know best, if the coding sequence of your vector insert should be OK; for example, if it came from an good and proven source, or, if there is a chance that there could be something wrong with the coding sequence: did you clone it yourself, for example, just by PCR? Then there is a chance that the subcloned sequence was wrong and not confirmed by sequencing and contains a stop codon or other mutations. But lets assume you have used a confirmed vector and sequence and the protein really induces some negative feedback, as you assume, I then could think of "in vivo footprint assays" to check if the promoter of your transgene is blocked by some proteins (blocking the known transcription factor binding sequences). This procedure would only be useful, if the RNA level is less than expected; so, I would check with a Northern blot the level of RNA of your transgene - perhaps, compared to any other cell, were the transgene worked, or compared to any other RNA within the cell. I guess, you expect tons of transgene RNA - and, for a toxin that could be a problem. Just a suggestion to think about a different (weaker) promoter for the transgene? But I guess it won't be easy, although very interesting, to find out what is really happening. I hope others have more ideas.

Hi,

I just want to add one more thing here. It is very important to check for the DNA contamination during the RT- PCR. By keeping the RNA for simple PCR we can check DNA contamination.

Hope it helps to conclude something

Hey Manjula, I have already done the DNA contam check..thnx!

Robert, I could check for the promoter blocking..thnx!

Hi Madhurima

Maybe your protein is not expressed in the level that can be detected by Elisa.The secondary structure of mRNA and the codon preference will affect the translation level. For toxin,there maybe extra control routs such as negative feedback by the toxin itself,as mentioned by Robert.So If your transgene have a tag fused to your protein coding sequence,you could try Immunoprecipitation or other affinity purification to try to enrich your target protein,

Or you can also try to add proteosome or lysosome(I am not sure whether this organelle exist in plants) inhibitors to check whether its level will be upregulated,this will help you to role out the possibility of rapid degradation after translation.

I agree with the above answers regarding codon usage efficiency. Since you see mRNA, inefficient translation might be the problem. One thing to point out though is since you are using RT-PCR, you may not be looking at full length mRNA. This could lead to a problem in interpretation (e.g. thinking that mRNA is OK). Since you are seeing an indication that the mRNA fragment you are amplifying is OK, other parts of the mRNA may not. You could run a Northern (RNA-gel blot) to see if full-length mRNA is present. If it is, then my bet is that you are looking at a translation problem (codon usage?). Good luck!

As your transgenic protein is foreign to the plant, it may be in a misfolded state and form aggregates. So you wont be finding traces of protein if you used the plant sap for ELISA. A western blot with pellet and supernatant of lysed plant sample would tell you whether this is the case or not.

Could you do any "functional" test with your putative toxin; for example, if it is a toxin against insects, you may check if it has a negative effect on insects. If it works, you only have a problem on the detection site.

Thanks Robert, I would try that out