Insertion of loxp/avoiding SINE - (Oct/22/2010 )
Apologies if this is not the right section for this question. I am trying to insert a single loxp (40bp with restriction site) into gene of my interest (9kb) which I have already subcloned into pBluescript. The position which is ideal for inserting loxp contains several unique restriction sites. When I analyze this region in UCSC genome browser it tells me that these restriction sites are located in Short interspersed nuclear elements (SINE). The ideal position for cloning is recognized as an Alu family repeat with the following description:
SW Score: 1043
I know that I have to avoid repeat elements for inserting loxp sequence into intronic region, in case that these elements might have a regulatory function which would jeopardize gene expression in tissues other than intended tissue. Since I have limited choice with selecting restriction enzyme, does anyone know that disrupting these repeat elements might have any effect on gene expression and whether insertion of a DNA sequence within these elements might be good idea?
Any suggestion would be appreciated. Thank you for your help.
this is very difficult question to answer.
Most Alu repeats play no (known) role.
However, there have been a few examples where the expression of the gene is actually regulated by Alu repeats, which contain transcription bind sites. It has even been observed that some Alu repeats are transcribe and these transcribe Alu repeat somehow regulate mRNA transcription of a gene.
Are these example rare oddities or are they hints that Alu repeats aren't quite as the silent parasite that they appear to be. Try looking journals for information. Maybe somebody has written something about Alu repeats and your gene. Failing that you will have to do an experiment and see if there is an effect.