I transformed a 6His tag fusion gene driven by AtUBQ10 promoter and expressed it in Arabidopsis. In T1 transgenic plants, first I used genotying to confirm they were real transgenic plants; In order to obtained trangenic lines with a decent transgene expression for study, I used western blotting against 6His antibody to check protein expression of this gene among T1 transgenic plants. Surprisingly, only ~10% transgenic lines showed strong protein expressions, the rest showed weak expressions. Is it often the case for you?
What could be the reason for the inconsistency of expressions among individuals??
Are there other genes that do not express when under a constitutive promoter?
Thank you!
Well, first, how many copies of your transgene in the line? Probably you have not used Southern analysis to determine the copy numbers of your T0 primary transgenic lines yet, only by using genotyping (with PCR?).
Transgene copy numbers influence overall gene expression results. If you have 3 unlinked transgenes in a T0 (on 3 different chromosomes, for example) transgenic line, after segregation, the derived plants in the T1 population will inherit different copy numbers of the transgene. Different copy number of transgenes might result in different gene expression profiles.
Even if your T0 primary transgenic line has only one single transgene insertion, the T1 population will have 3 different genotypes (+/+, +/- and -/-) for the transgene. The gene product can be different from +/+ and +/- plants. This has been observed and reported using GUS gene.
Hii Yuan, Many factors are worth considering here. The expression of introduced gene can be temporally as well as spatially regulated. Finding a stable transgene (with same bahaviour over generations) is not that easy. As we know that every individual plant carries a different transformation event, so its behaviour will be different. As Prof. Yuan-Yue described, you need to find out copy number of the lines, take care of this factor. Also remember 35S has also been reported for differentially regulation. Please see the litearute, same might be case with uniquitin. You may need to find homozygous line over a cycle of selections (+/+, +/- and -/-) in order to obtain a stable transgene. Good luck
Hello Yuan, what's been said above is relevant but I would like to add that the location of the insertion in the genome is, in my opinion, the real big driver of gene expression level. Some areas are heterochromatine rich others euchromatine rich which would result in the same single insertion homozygous transgene being expressed at varying level (from undetectable to very high level). Typically we screen 15-25 plants in our lab and keep three lines that have a good expression level.
Thanks everyone for all your answer!
For copy number, I checked segregation ratio for T2 generation plants by growing young seedlings on selective medium. Most are single copy, but their expressions in T1 generation still varies dramatically. Perhaps the position effect does cause a big variation in gene expression.
I am also curious about how different copies get integrated into an individual plant? Or it's just randomly T-DNA gene insertion.
Thank you!
@ Yuan,
1. If you designate 'T1' (not 'T0') as the primary transformants, then the vary gene expression among these T1 primary transgenic lines are most likely due to (1) position effect and (2) transgene copy numbers. Multiple copy transgene insertion tend to cause transgene silencing. This is well documented.
There are also other reasons, such as the transferred T-DNA is truncated (not intact), and the truncated transgenes will lost its function (no expression).
2. The copy numbers of T-DNA insertion can be different among the primary transgenic lines. I have seen 0, 1, 2, 3, 4 or >5 copies in a transgenic line. How different copies of transgenes get into an individual plant is unclear.
Hi Allah Bakhsh,
Some genes are temporally or spatially regulated in planta. However, all the transgene in T1 progeny are with the same promoter and the same gene. Hence, under normal condition, these genes should all subject to the same temporally and spatially regulation (if any).
See attachment: "Temporal and spatial control of gene expression in horticultural crops"
Dear Yuan-Yue, Thank you for sending the article. I will go through it. Your point remains valid, I just wanted to draw the attention of fellow researcher to a particular point that have been documented. The ability of 35S promoter being costitutively expressing one has been questioned, but I did not read anything like this about ubiquitin promoter. As a reference
https://www.ncbi.nlm.nih.gov/pubmed/12369622
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