Hi...

The negative GUS staining could be due to follow reasons:

1- Insertion of T-DNA in a low expressed region of chromossome (i think that this not is your case, because probably you screened several independent transgenic lines...)

2- Very low expression of your gene (the expression is driven by the promoter of your gene or another?)

3- Mutations in GUS or your cloned gene (did you sequenced your construct?), or T-DNA is not in your plants (i think that you could do some PCR reactions with genomic DNA to confirm the insertion (use GUS primers, for example).

But in your case, i think that the absense of staining is due to presence of a stop codon in your cloned gene, stopping translation. Do another primer (only reverse) and remove the stop codon of your gene. Be carefull with the reading frame, because your gene and the gene reporter GUS must "in frame". If necessary, you can add 1 or 2 bases after your gene to keep in frame.

Good luck

Cleverson Matiolli

Hi,

If you say its not expressing check the reading frame and sequence of your construct that you have used to make transgenic.

What selection marker you have used to screen the transgenic line, because some selection markers like hygromycin are not that efficient in bleaching out seedlings, meaning even non-transgenic also grows and look green, unless you allow for more no. of days at least 2 weeks till they develop true leaves.

If you really are confident that what ever the material you have screened is really transgenic, then you identify at least five independent transgenic lines. Because many at times because of positional effect transgene may not express.

Also it depends on type of gene, if the gene inducible by some kind of stimulus then you dont see any expression with out that.

If all the things are fine, then its possible that very weekly expressed, in that case you better go for quantification assay by using MUG.

Good luck,

Sreeram gangappa

-Resequence the construct.

-Confirm that you are analyzing transgenic events and not escapes.

-Use proper controls (+ve and -ve ) in your GUS assay. Check X-Gluc or your assay buffer with +ve controls. If possible use a fresh lot.

-What is the promoter? if it is a weak promoter, use fluorescence based methods to detect low activity.

To get GUS expression using pMDC139, you need a functional fusion protein. In your case, I think, protein is not functional because of the presence of native stop condon. Dual 35S promoter is strong enough to drive the expression of the reporter gene even if the expression of your gene is weak. After developing a functional fusion protein, if you don't get the expression, then you have to think about growth stage or tissue specific expression of your gene of interest.

Good luck!

Cleverson Matiolli:

But in your case, i think that the absense of staining is due to presence of a stop codon in your cloned gene, stopping translation. Do another primer (only reverse) and remove the stop codon of your gene. Be carefull with the reading frame, because your gene and the gene reporter GUS must "in frame". If necessary, you can add 1 or 2 bases after your gene to keep in frame.

Hi,

If the expressions of your transgene and GUS are driven by the common promoter then definately stop codon is the matter to think. But still you can confirm the presence of gene by seperate PCR for transgene and GUS. If it shows the presence of both gene then you can focus to solve the stop codon issue.

And if expression of both genes are driven by seperate promoter then stop codon of your gene will not be the cause. In this case, there is the chance that T-DNA is intrgrated in low expressing regions like heterocromatin region as suggested by Cleverson Matiolli.

Many times PCR shows presence of gene but GUS assay gives negative results. Even this happens when construct is checked by sequencing, ORF finding and virtual translation.

If your construct is OK for sequences, promoter and ORF; plants are showing presence of gene and GUS by PCR then you can try qPCR to check the expression of GUS.

Best luck,

Swapnil

Hi sir, what i suggest is that after trying all above options you once confirm the method used for gus expression, you can include positive control! Your gene might have expressed but in less amount. Take observations under confocal microscope so that even small amount of expresion is visible!

I wouldn't test too many options. The best would be to clone it again without the stop codon, which is likely to be the problem (since you're using the gateway system, it shouldn't take too long) and get new transgenics (for Arabidopsis it's not too much of a big deal neither). Then you'll see.

The other problem I could think of is your GUS assay. Do you have a GUS control (i.e. a plant expressing GUS constitutively, or whatever)? Maybe the solutions you're using have something wrong. Unless you're totally used to GUS staining, in which case it shouldn't be the problem ;-)