I'm trying to make a construct that I will use with TALENS to put a fluor at the AAVS1 locus.
I have the AAVS1 left and right homolgy arms (HA's), but from a previous construct, there is an insulator/blocker element 3' to the left homology arm. After that is a promoter and a fluor, followed by a pA and then the right homology arm.
For my new construct, I'm making a differentiation reporter by putting in a gene-specific enhancer sequence followed by a beta-globin promoter and fluor. I chose the beta-globin promoter because I want the expression of my fluor at low levels until my gene is expressed. I was going to put the construct together as:
backbone--HA(L)--insulator/blocker?--gene-specific enhancer--beta-globin promoter--fluor--WPRE--BGHpA--HA(R)--backbone
But when I blasted the insulator sequence, it came back as actually being part of the chicken beta-globin gene.
From everything I've read, it seems the insulator sequence is part of the AAVS1 DNAse1 hypersensitivity region and used to assist with maintaining an open chromatin structure.
My questions are: does anyone know what the insulator being present does to the gene expression if you are putting a promoter-driven sequence at the AAVS1 locus? Do you think it will affect the effectiveness of my gene-specific enhancer for promoting my fluor expression? Can I take it out or will it affect the openness of the AAVS1 locus? Should I put my sequence in backwards between the two HA's just in case?
Please help!
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