I generated several transgenic Arabidopsis lines that contain a T-DNA in which two transgenes (Gene A and Gene B: see attached figure for more details) are linked. Each transgene has its own promoter and terminator. When I measured the relative expression of each transgene in the transgene lines, I found that the expression of each transgene was highly variable between lines which is probably due to the integration of the transgenic cassette in different genomic position. Most importantly, there was a significant difference between the expression of Gene A and Gene B in most of the lines whereas equal expression was found in one line. As both gene experience the same position effect,
1. Why was there a difference in expression between Gene A and Gene B. I guess this probably due to the difference in promoter strength. If this is due to promoter strength, then why in one line they are expressing equally?
2. Is there any other explanation besides those I mentioned above?
3. How should I test whether the difference in expression of Gene A and Gene B in the same line is a promoter effect?
Differences in integration sites will put your cassette in a different regulatory context in each transgenic line. Since your Gene A and Gene B face in opposite directions, they can be differently regulated by local regulatory elements in the genome. Also, you can get methylation of your cassette after integration, which will down-regulate expression. Depending on the integration method, you can also get truncation of your cassette, which will reduce expression of just Gene A or just Gene B or both. Map and sequence your inserted cassettes to get a better explanation (if it matters). Or just screen more lines until you get several with the expression pattern you need for your project.
Expression of any gene depends on the set of many factors and cellular processes. This dependence not only on the strength of the promoter, the presence of other regulatory sequences, the location of the transgene after transformation, but also on the functional load carried by the gene itself in the plant (whether it needs the cells, in what processes participates, how important this process is for the cell, the amount protein, necessary to perform a certain function), are there factors inhibiting the work of this particular gene at different stages of expression ... Hence the reasons for the different efficiency of the formation of transcripts. If to answer the last question, I would advise to swap places and make a new transformation. In any case, you will learn much more about your genes.
Differences in integration sites will put your cassette in a different regulatory context in each transgenic line. Since your Gene A and Gene B face in opposite directions, they can be differently regulated by local regulatory elements in the genome. Also, you can get methylation of your cassette after integration, which will down-regulate expression. Depending on the integration method, you can also get truncation of your cassette, which will reduce expression of just Gene A or just Gene B or both. Map and sequence your inserted cassettes to get a better explanation (if it matters). Or just screen more lines until you get several with the expression pattern you need for your project.
Integration could affect A and B differently independent of the promoters or enhancers. Imagine that there is another gene and transcript being trasncribed from an endogenous flanking gene on the right and reading into your cassette. This would create an anti-sense transcript for gene A but not gene B, since that is in the same orientation. Thus the anti-sense for A could suppress translation or promote RNA degradation of gene A but have no effect on gene B since it is coming off the other strand.
Just one of many possibilities.
From the above I think Shu Wei is closer to the problem. My guess is that you do not correct for PCR efficiency between the 2 products. ie if you amplify eual amounts of the transposon in a tube by either primer sets do you get equal amounts? . From the graph is seems that high expressors are more or less equal. I think that either you have a lot of error in measuring low values or you just measure background.
All above statement are worth considering, I just would like to add, do you know the copy number of your genes A and B. Though they are in same cassette, but as someone also mentioned here, you might end up with genomic rearragements, different integrations/insertions pattern etc, therefore, if it is the case, copy number can affect expression of your genes.
Allah Bakhsh's suggestions seems to have a logic. It will be useful to know copy number of each gene transferred.
Quite an intelligent Q & important one in view of the delicate problem.
I agree with what Oxana Karpova & Shu Wei have said.
Also take care of what Allah Baksh has warned regarding Genomic re-arrangements affecting the copy number & expression of genes.
Best of luck in redoing your expts & confirming the results obtained.
I agree with all above,
I also presume the the gene A and gene B are two different genes and sure they have their own information on time, space and quantum specific expression, so the difference in the expression. All such have to be compared with the control including the controls like individual gene cassette.
- Badges
- Science topic