Hi all,
I have very little experience in this. I would like to develop a reporter assay, by fusing GFP to the response element of a particular nuclear receptor. I wanted to accomplish this using either CRISPR or Tol2, and was wondering if it is necessary to generate a fusion construct consisting in GFP and a proximal promoter, or if it is possible to to insert only a GFP sequence upstream of the regulatory element using the CRISPR-Cas system? I apologize if it seems silly.
Hi Michael,
In both ways you can get your readout. However you can expect differences depending on whether the GFP, together with the proper promoter, is inserted via Tol2 or the GFP is precisely inserted in the locus of your GOI.
In the case of using CRISPR, as you imply, you can choose to insert the GFP in frame with your GOI, creating a fusion, or inserting it in the locus but being transcribed independently.
The first has been described in zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/25740433) and we have also done it in medaka fish (http://www.ncbi.nlm.nih.gov/pubmed/25909470).
I hope that this helps you.
Best,
Juan
Hi Michael,
In both ways you can get your readout. However you can expect differences depending on whether the GFP, together with the proper promoter, is inserted via Tol2 or the GFP is precisely inserted in the locus of your GOI.
In the case of using CRISPR, as you imply, you can choose to insert the GFP in frame with your GOI, creating a fusion, or inserting it in the locus but being transcribed independently.
The first has been described in zebrafish (http://www.ncbi.nlm.nih.gov/pubmed/25740433) and we have also done it in medaka fish (http://www.ncbi.nlm.nih.gov/pubmed/25909470).
I hope that this helps you.
Best,
Juan
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