Yes. There are many post-transcriptional mechanisms to control de gene expression. interference RNA is very common in plants, for instance.

Hi Juliano,

Can you please suggest me, how to elucidate or establish the  kind of  repressor mechanism related to my case study?

Thanks

You said that you have obtained different transgenic lines. Have you checked to see how many copies of transgenes in the different transgenic lines. It is well documented that multiple copies of transgenes tend to cause gene silencing. If your transgenic lines have multiple copies of transgenes, then you probably encountered gene silencing.

Therefore, we usually try to obtain as many transgenic lines as possible. Then we screen for lines which contain single-copy transgene. After that, screen single-copy lines with the best performance. Due to 'position effect', some single-copy lines will out-perform the other single-copy lines.

Also, you can consider to do a Northern analysis to see whether the transcripts are increased as compare to the wild type.

Hello Yuan

I did check for copy number of transgenic lines using southern blotting and lines only having the  single-copy insertion were selected for analysis. I took around 15 single-copy lines but none of the lines were showing transcript or  protein accumulation. 

Is it worth doing northern blotting? As RT-PCR analysis did not show increase in the transcript level in any of the lines.

Thank you

Like Juliano said, RNA interference is rather common. It is often induced by high expression and/or by "miraculous aberrant RNA". Nobody really knows what that is, probably uncapped or not polyadenylated. Intron-containing genes may be less prone to RNAi than intron-less genes If you used a cDNA you may try a genomic copy. You may also try the endogenous 3' UTR and polyA site.

I am not sure if it is worth the trouble but you may try to check if the problem is really RNAi by doing a Northern on small RNAs. If you find 21mers that hybridise to your gene sequence you know what the problem is - unfortunately it does not make the problem go away :-(

Hi Roshan,

As you have already mentioned that your plasmid may not work. I am worried about that too. How are you going to take care of this part? Had you checked your plasmid carefully after you build it and before you did the transformation?

Hi Yauan

I took all the care  at the time of development of over-expression construct and even at the transformation steps , possibility of getting wrong is very low as  two different independently developed  construct  for two different gene of same gene family were used for transformation and none of the lines from two independent constructs  showing accumulation of transcript

Hi Yauan,

Have you take into consideration that the promoter you used to drive your transgene expression might not be active in your cell? Or have you try other construct with different promoter?

I might be wrong as I never work with plant.

Roshan,

I think that Wen Hao's question was meant for you to answer......

Hi Roshan,

How did you arrange your genes in the plasmid? The orientations of two genes in the vector?

For example, if 35S::gene 1 -->

Hi Roshan,

I'll jump in with a couple thoughts/questions.  First, you have not mentioned selection of your transfected cells.  Is there a selectable marker on your construct, and is the selection working well?  If so, this might argue against a positional effect that is leading to silencing, especially if you are choosing single-copy integrations.

Second, along the line of what Wen was asking, are you confident in the robustness of your constitutive promoter?  On the other end, are you confident in the quality of the 3' UTR (i.e. is this a standard 3' UTR used for plant constructs)?  Finally, does your construct contain a Kozak sequence?  A lack of Kozak sequence would not necessarily explain why there is no increase in transcript abundance, but it would certainly explain a lack of translation.

Given that both of your independent constructs are failing to generate transcript or protein, my best guess is that the issue is with the vector rather than the genes themselves.  So I would look closely at the promoter, 3' UTR, terminator and Kozak sequence.  If the 3' UTR is not a standard 3' UTR used in plant expression constructs, be very sure that there is a good polyadenylation site and that there are no instability elements (e.g. AREs).

Good luck!

-Jason

Hi Jason,

The binary vector which I have used for over-expression construct  is gateway binary vector "pEG100", which is one of the well establish vector used in plant system and this vector system to have the selection marker gene to select the recombinant plants. In my case selection was quite clear between recombinant and non-recombinant plants. Apart from single copy lines I also checked the transgenic lines having multiple copies and sadly result was same. I have also made construct for other genes using same vector system, and all the constructs were working quite good.

Is there is any way to  check the degradation of  transcript above the substantial level due to over-expression in my transgenic lines as all the lines having the transcript similar to wild type and vector control?

Thank you 

HI Yaun

I have only used single gene to develop my construct. Earlier, I mean to say that two different constructs (plasmid) were developed using different members of same gene family and both the constructs or plasmids are independent to eachother.

Thank you

Did you generate your construct using cDNA for your gene of interest?  If so, the transgene presumably contains its own endogenous 3' UTR.  I'm guessing this is the case since the pEG100 vector does not include a 3' UTR.  I think you need to consider whether there are any known instability elements in your gene, especially in the 3' UTR.  AU-rich elements (AREs) are perhaps the best studied.  Unfortunately, the databases for ARE-containing transcripts are all for vertebrates, and I can't find any for plants.  An exosome-sensitivity element was identified in 3' UTRs of certain Drosophila transcripts, but I don't think this has been studied in any other models.  There is also evidence in yeast that some transcripts are slow in their nuclear export, and this slowness sensitizes them to exosomal degradation.  However, in this case I don't think any element has been identified that slows export.  These are some other explanations that could account for transcript instability off the top of my head, but unless they have been specifically examined for your gene, it would probably be difficult to predict such effects (with the possible exception of the AREs, which have a well defined sequence).  The presence of AREs seems to correlate with the potency of the gene product.  For example, cyclins, c-myc and c-fos contain AREs, and the resulting transcript destabilization appears to be a safety mechanism to prevent their overexpression. If your gene is detrimental when overexpressed, it may follow that an instability element could be present.

This brings me to my next point, if your gene is detrimental when overexpressed, there may be a selective pressure against your transgene's expression.  Perhaps only the cells that silence you transgene are able to grow.  Has this gene been overexpressed previously?

I think it would be ideal to perform RT-qPCR with an assay that is specific for your transgene.  If the construct is based on a cDNA, which includes the endogenous 3' UTR, this could be difficult without some sort of silent mutation (i.e. in a UTR, or a synonymous coding mutation).

Hi Jason,

Thanks for providing the information  but in my case I have used only the CDS region. As I earlier mentioned, I did check the expression of transgene using qRT-PCR but none of the lines were showing transcript accumulation  as compare to wild type and vector control 

Thank you

Hi Roshan,

If you are really interested why your construct is not expressed, you may try nuclear run-on transcription (not really sure how well this is established in plants though).

This is rather old-fashioned but it will tell you if your gene is transcribed (even if you do not see transcripts in Northern or qRT). Use a low and a high expressed endogenous gene as a control to get an estimate on the level of transcription. If you are intersted I could give a few hints on the method.

In some cases, when a protein is overexpressed, it is displaced from its natural complex and is degraded. In principle, this can happen when some mRNAs are overexpressed, although I have not seen it in the literature. In addition to the runon, proposed earlier, you can try blocking transcription by actinomycin D and determine the actual half life of the mRNA. 

If your transgene does not have any 3' UTR or polyA site, this could explain your lack of expression.  Where did you get the CDS from, if not from cDNA?  

Is your qPCR assay specific for the transgene, or could it also detect the endogenous gene?  The fact that your qPCR is showing similar levels in your expression cells as in your empty vector controls suggests that your assay does target the endogenous as well as the transgene.  My point was that you could try to design an assay that only detects the transgene.

Hi Nellen

Can you plz suggest  some protocols and hints regarding nuclear run-on transcription. I will really appreciate if you provide some methods without the use of radiolabelled substrate

Thank you 

oups! I have never done it without radiolabel - no idea if it works with sufficient sensitivity as non-radioactive!

The method is described in the two rather old attached papers. What I do not know is how easy/difficult it is to get live nuclei out of plant cells. Maybe someone else can help with that.

Hi Jason

I have tried to over-express one of the endogenous gene of the same plant under constitutive promoter. So qRT-PCR can not differentiate between the over-expressed and endogenous gene. Therefore, we can only quantify the expression with respect to vector control lines.

I have amplified the CDS directly form the cDNA using gene specific primers.The binary vector which I have used contain "ocs polyadenylation" signal at the 3' end.

If, RT-PCR analysis did not show increase in the transcript level in any of the lines, it's not ncessery to do Northern.

There is many possibilities :

-your construction is  not correct for raisons that Yuan mention; or your gene dont have poly A tail that increase stability of the transcript, or your protein is not in frame in your construct.

-the promoter that drive your gene is not compatible with you species. Which species you used ?

- effects of gene silencing, impossible to have silencing gene in 15 transgenic plants.

So, chek your construct and your vector.

Zahra

-

Hi Roshan,  have you tried to transform a control construct, expressing a reporter gene (GFP, dsRed, beta-gal, etc, whatevr you use in plants) in your plasmid? If not, I would certainly do it to check if the  regulatory sequences within the plasmid function properly.

Hi Daria,

Yes, I transformed the construct having my gene of interest in fusion with YFP, and I will be doing qRT-PCR and western blotting to check the efficacy of the constructs

Thank you

I guess what Daria means is not a fusion but just GFP or YFP in between your promoter and terminator (best could be to even leave the 5' and 3' UTR of your gene in and just exchange the coding sequence)