I just obtained homozygous line of one of my overexpression lines. I chose this line for propagation because its expression was highest among other transgenic plants when I use western blotting to examine their expression in T1 generation.
However, recently when I did western blotting again in the T4 generation of my transgene using young leaf tissue, this protein was barely show up in the western blot. Then I tried really young seedlings, this protein can express.
How it possible that the protein lost its expression in later developmental stages or in later generations? This T-DNA insertion is a quite conventional method to generate transgenic plants. Would this gene get silenced in future generation?? Should I care if the gene not express very well in later stages, but does express in seedling stage?
One of the possible causes is methylation. Methylation causes partially gene silencing or completely gene silencing. Methylation of your transgene or the promoter region could have occurred at later generations. Gene silencing occurs at later generations has been reported.
Hi Yuan,
Here is an example:
Please see the attached paper (PNAS 2002): "Tissue-specific silencing of a transgene in rice"
Below are some summaries taken from its Abstract of the paper:
(1) Promoter (tungro bacilliform virus promoter) of transgene became gradually methylated, and gene activity was lost concomitantly.
(2) In further generations of homozygous offspring the methylation spread into the transcribed region and gene activity was completely repressed
(3) The gene activity could be reestablished by treatment with 5-azacytidine
Thank you so much for the reply and the paper attached. I use UBQ10 promoter to induce my gene expression. I am screening more transgenic plants now.
Do you think if I screen more homozygous lines, some may turn out good. I searched previous publications on UBQ10 promoter, it can be used to examine Ca+ changes in plants.
Hi Yuan,
1. For your #2 question: Can the protein lost its expression in later developmental stages?
Yes, it also has been reported. Please see the attached paper:
"Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants"
2. For your #3 question: Should I care if the gene not express very well in later stages, but does express in seedling stage?
Yes, you should care. That is the purpose to transform a plant with a gene-of-interest: to get the transgene expressed in the plant. A good transgenic line should be (1) fertile (not sterile), able to produce a good amount of seeds, (2) the transgene can transmit into the future generations, (3) the transgene can actively express (in the parental and future generations)
Hi Yuan,
Here is another good example of transgene losing its activity after several generations:
From the Salk T-DNA insertion lines, occasionally you will find the lost of their kanamycin resistance ability after several generations.
Here is the answer to one of the Frequently Ask Questions from their website:
http://signal.salk.edu/tdna_FAQs.html
8. Q: What is the plant selectable marker used for the transformation?
A: The marker is NTPII (kanamycin resistance). However after several generations of growth, some of the lines show silencing of this gene. Thus, it is not unusual for a mutant line to not express the drug resistance phenotype.
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