I'm currently studying a family with a huge duplication which encompasses a tissue-specific enhancer element. Therefore I want to investigate the effects of such duplication.
I was wondering whether it is possible to use a luciferase assay to do so. I could make a construct with a tandem duplication of the enhancer element, insert into a plasmid and see if there is any difference between the contruct with only one copy of the enhancer compared to the one with two copies.
I also brainstormed a few other strategies, like a CRISPR;Cas9 assay to remove the duplication, but since it is a 400kb duplication, I guess it's not that trivial.
Thanks a lot!!
Sofia Ramos
You can insert multiple enhancers within cell lines that a similar to your tissue specificity. In my paper (linked in this comment) we measured the effects of various enhancers and silencers within a particular gene of interest. Article The regulatory control of Cebpa enhancers and silencers in t...
I agree to Shawns comment. Additionally see alternative literature dealing with a comparable issue (CA repeats eNOS or ESE/ISE Tissue Factor splicing).
See:
a) Lorenz et al., FASEB J. 2007; 21(7):1556-64. (Article Alternative splicing in intron 13 of the human eNOS gene: A ...
)b) Tardos et al., J Thromb Haemost. 2008; 6(5):877-84. doi: 10.1111/j.1538-7836.2008.02946.x.
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