Hellow fellow academics
I am currently in a dilemma and I would really appreciate some suggestions/guidance on the matter.
Situation:
I have overexpressed my gene of interest (GOI) from wheat in Arabidopsis using the floral dip method and with strict screening on MS-Hygromycin, obtained my T3 transgenics. Now the problem is that while the selection, on the media has been successful, I am not able to get a band of my GOI on agarose gel after doing semi- RT-PCR. Initially, I thought that maybe my overexpression was unsuccessful so I took the T3 seeds to screen again on the media, but, the result was the same; the overexpression was successful and met the segregation ratio requirement of 100% germination. As this is my first time working with transgenics, please enlighten me on where I could be going wrong.
Please advise. Thank you for your time in advance.
Dee
Keep in mind that the selection on hyg and the expression of your GOI are two different things. The selection is based on the hyg gene in your T3s. The expression of this gene is controlled by a strong constitutive promoter.
The expression of your GOI is controlled by the promoter you cloned it next to. What promoter are you using? If it is not something expressed in the tissue sample and growth stage you are sampling then you won't get anything with your RT expression analysis.
Amy Klocko Thank you so much for your response. I have used a 35S promoter to clone my GOI and used leaves from the adult Arabidopsis plant as my tissue. Could you please direct me to a read and/or suggest some troubleshooting mechanisms which I could employ to identify the cause of the problem? Thank you so much again for giving me your precious time.
Ok, that helps a lot. So, if your GOI is controlled by the 35s promoter there are a few possible explanations for the lack of expression in your assay.
It is possible (but not common) for an overexpressed gene to be toxic and thus silenced by your plant.
It is likely that your expression assay is not working properly and could use more controls. Here is what I suggest.
1. genotype your T3 plants for the 35S:GOI fusion construct (F primer in 35S, R primer in GOI), this is standard end-point PCR on gDNA
2. use a housekeeping gene as a control for your RT assay (actin is a good choice) in general
3. re-try the RT of your GOI, use a kit for the RNA extraction, be sure to DNAse treat your RNA, and use oligo-dT for the cDNA synthesis reaction.
hope this helps!
Amy Klocko Thank you so much for your guidance. It helped a lot and I was able to continue my intended experiments successfully.
That's great! Thanks for the update. What ended up being the issue with the RT work?
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