6 replies to this topic

[gyma]

hello everyone, I am now trying to cut the shRNA sequence from vector A and insert it to vector B. Unfortunately, I dont have a good enzyme site at the 5' of the shRNA. I have to cut about 70 bp upstream of the shRNA. I want to know whether this extra 70 bp will make the shRNA in the new vector ineffective. I am also confused about where and how the shRNA sequence is recognized. please help, thank you.

[gyma]

nobody knows? what is the optimal distance between a promoter and a transcription starting site when constructing a vector? I guess different kind of promoters should have preferred distances, or not?
From NCBI, it is easy to know where the TSS is because they provide the full mRNA sequence. But how do they know that? 3' end might be recognized by the presence of a poly-A tail at least. How about the 5' end? Is it a too basic question to answer?

[bob1]

I don't know the answer with respect to shRNA (which is why I didn't answer before) but in normal plasmids the promoter distance isn't too critical, but I don't know how far is too far.

Edited to add: a quick search indicates that 100 bp between promoter and gene sequence isn't a problem for some widely used plasmids (e.g. pcDNA3-eGFP).

[gyma]

bob1, on 15 October 2012 - 12:05 AM, said:

I don't know the answer with respect to shRNA (which is why I didn't answer before) but in normal plasmids the promoter distance isn't too critical, but I don't know how far is too far.

Edited to add: a quick search indicates that 100 bp between promoter and gene sequence isn't a problem for some widely used plasmids (e.g. pcDNA3-eGFP).

Thank you bob1. I checked several shRNA vectors and the distance between U6 and shRNA sequence seems to be close. An extra length might at least reduce the transcription efficiency of the shRNA even if other aspects are not affected. Thats my thought. Thanks anyway.

[bob1]

Those were my thoughts too.  Unfortunately, I have no idea for shRNA promoters.  I know for RNA transcription using SP6 or T7 the promoter is right at and often overlapping the gene start site.

[gyma]

bob1, on 16 October 2012 - 02:54 PM, said:

Those were my thoughts too.  Unfortunately, I have no idea for shRNA promoters.  I know for RNA transcription using SP6 or T7 the promoter is right at and often overlapping the gene start site.

thank you for your help.

[gyma]

bob1, on 16 October 2012 - 02:54 PM, said:

Those were my thoughts too.  Unfortunately, I have no idea for shRNA promoters.  I know for RNA transcription using SP6 or T7 the promoter is right at and often overlapping the gene start site.

thank you for your help.