RNA activation but not RNAi? - (Jul/07/2006 )

Let me introduce our idea firstly.

my supervisor's idea is that RNA can active the original express of DNA . In his theory , non-coding RNA (maybe any other types of small nRNA) is the basic trans-acting factor of transcription. Some maybe small RNA combined with the folded DNA, then after the reaction of DNA-RNA-protein, the DNA become unfoldedand then transcripted. But it is very hard to judge whether it is right or wrong because there is no evidence about it.

So , he designed our job on the reported experiment that if insert ORF2 of LINE-1 (the biggest family of noncoding DNA,ORF2 is about 3900bp length,and can't express protein ) into the pEGFP, the L1 will decrease the express of GFP.The inhabitation was happened from the level of transcription by Northern-Bloting Result. And the inhabitation was related to the length of the L1 sequence but not to the specific sequence itself.

Based on that experiment, firstly, we divided the ORF2 to three parts: ORF2a,2b,2c and then recombined a new plasmid by inserting each of the fraction part(about 1300bp)into pcDNA3.1.what we want to know is whether pcDNA3.1ORF2a,b,c RNA can uprise the GFP express of pEGFPORF2. And we use the pcDNAlacZa and pEGFPlacZ which of them both have the same length of ORF2a/b/c and ORF2 as the contrast.

Secondly we transfected the pcDNAORF2a into Hela, selectived by G418.After 1 month,we got the stable pcDNAORF2a,the same to 2b or 2c.

Thirdly,we transiently transfected pEGFPORF2 into stable pcDNA3.1ORF2a Hela, pcDNA3.1lacZaHela, pcDNA3.1Hela, Hela.

then,we observed the GFP express after 24.48,96hours by fluoroscope .Confusingly, the fluorescence intension is 2a>lacza>pcdna3.1>Hela .Undoubtly, the stable transfected cell increase the expression of GFP,but we don't understand why all of them even pcDNA3.1 can active the GFP express of pEGFPORF2.

I WANT TO GET SOME SUGGESTION OR ADJUDGEMENT ABOUT OUR JOB.IS there any flaws in our DESIGN? Any suggestion is uesful to me , my topic maybe so long that i hope it will not boring u before you read the end of it.

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There are reports (from a conference) that small RNAs targeting promoter region can activate gene transcription in a sequence-specific manner.

Probably transcripts from Line1 can form some stem-loop structure which can be processed by dicer to small dsRNAs. If these dsRNAs have target site on the regulatory region of the vector expressed gene, they may activate transcription from the vector.

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A number of key information were not provided which made it doifficult to comment. For example, the fold of difference of EGFP expression among various clones, more importantly, are the difference statistically significant? Also, I would ask that you look into transfection efficiency of individual Hela cell clones more carefully, as you are study individual colonies rather than the original Hela line, and I certainly would ask if these individual colonies have similar transfection efficiency among themselves and the original line by the particular transfection method that you have used. Have you done a co-transfection study to evaulate if that is the case, and if you did, what would be the proper promoter to use. I hope these comments help you on these minor details first before coming to more complicated issue as you have had in mind. Good luck.

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reply to pcrman:

very glad for your reply! Still i did't find any papers about RNA activation. So i would appreciate

getting some details about the reports from the conference you mentioned about. Thank you very

much!

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reply to genehunter-1:

thank you very for your question and your suggestion! i want to give some details of my
experiment.
Firstly, from the result the GFP expression have a significantly statistical discrepancy .
Secondly,we also transfect the pEGFP-C1 into different cell lines as the contrast and we find there is no significantly different between them.But i have a question if the pEGFP-C1 have expressed to the utmost that we can't see the difference.This is same question to your suggestion :whether they have the similar transfection efficiency.I am just a graduate student and only did one year research .So i want to know if there is some changes for the cell line after the stable selective:for example the cycle time of cell proliferate,and the rush time of transfection .
Thirdly: we have did co-transfection also we find pcDNA3.1ORF2a/b/c will enhance the express of pEGFP, but the discrepancy did't significantly.I am sorry i did't get meaning of " what would be the proper promoter to use" .
Thanks for your good-hearted suggestion again!

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You can find the abstract in 2005 AACR annual meeting proceedings.

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1. Good to know that there is a significant difference.
2. There are several possibilities.
a. In principal, individual colonies can have different response to the same transfection agent, although I am not sure anyone has seriously look into this issue. No one has success of 100% transfection efficiency for Hela cells. Why cannot you transafect all of them? You could say that is because cells are in different stage of cell cycle, but other factors may also contribute to this. I would not think Hela cells are homogeneous after so many passages.
b. You generated colonies by introducing genes to cell via a transfection method, and then introduced another test gene to these colonies using the same method. There will be some bias that cell colonies maybe in favor of the same transfection process than the original Hela cell population. I would use a totally different transfection method (electroporation, for example) for your reporter and reference plasmid (see late).

3. People use a reference reporter gene (beta-galactosidase, for example) co-transfected with the target plasmid together and assay for the reference and target gene expression as a way to normalize their gene expression results. This is to rule out variation due to diffrerece of transfection efficiency between experiments. You certainly will not want to use pCMV-gal as it carries the same promoter that you are studying, maybe pRSV-gal, but you have to test it out.

Since you have failed to demonstrate difference by transient transfection, working on stable lines using another transfection method will the next choice. The best way would be to do stable transfection for the interested gene sequence and the reporter gene, preferably using an inducible promoter (Tec-On, for example) for the interested gene. Of course that would take you another month or two.

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Thank you very much!I will search it online*_^ thanks a lot!

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2005 AACR annual meeting proceedings - I cannot open the link on the AACR homepage.
There is sth called the AACR Education Book with about 100 articles of the 2005 meeting. In which article can I find sth about RNA activation?

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