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I want to change a pre-miRNA sequence (in my case the pre-miRNA is encoding in a 3'UTR of a gene) and then put it in a lentivirus to see if it is still processed.

After modification (permutation of ten regions of ~20 nt) which kind of newly appeared pattern I've to be careful about? I do not want to disturb the gene in which the miRNA is encoded

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  • $\begingroup$ I don't understand what you're doing. It seems like you're altering 10 pre-miRNA genes in the 3'UTR of a protein-encoding gene. What do you mean by "which kind of newly appeared pattern I've to be careful about"? Are you wanting to assure that regulation of the protein-encoding gene is unaffected? Why? Are you going to knock out the genomic locus and rescue with your viral copy? $\endgroup$ – KAM Jan 16 '12 at 16:01
  • $\begingroup$ Yes I want to assur that regulation of the protein-encoding gene is un affected. I m gonna pertub the secondary structure of all pre mirna to delete the drosha processing. After that i m gonna put the region into a lentivirus and infect cells, and then check if the mirna are no more expressed. $\endgroup$ – Nicolas Rosewick Jan 16 '12 at 20:04
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If your goal is to avoid regulatory elements, I would not believe any prediction program, since new regulatory elements are found every day. The best way would be empirical and just clone and infect with a mutated 3'UTR and see if your gene's regulation is perturbed. You could at least swap the entire 3'UTR with that from another gene to see if there are even any gene regulatory elements in the 3'UTR.

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