I have conducted a study using Tol2 Gateway system to study human promoter in zebrafish. As per literature, the system should have worked, but it didn't. The zebrafish positive control was positive, but the human was not. What would be the possible reasons for it?
Many thanks!
Hi Monika,
One problem with the Tol2 system might be positional effects due to the random integration.
Maybe "PhiC31 integrase" system works better since you would use specifiic landing sites in the genome.
Site-directed zebrafish transgenesis into single landing sites with the phiC31 integrase system.
Mosimann C. et al. ; Dev. Dyn. ; 2013 (please find attached)
Easiest explanation would be that the human promoters do not have activity, either none at all or they do not work in fish. Promoter sequences are quite divergent between fish and mammals.
For trouble shooting:
- If you used gateway for cloning check that all pieces are where they should be by sequencing. Gateway is prone to give false clones if it does not work for any reason.
- Does your human promoter have a polymerase binding site? You might have cloned only an enhancer and need a minimal promoter. There are several used in zebrafish we used this one sucessfully: http://www.addgene.org/22453/
There might be a position effect as Martin mentions. But you can exclude this by looking at several lines form the same construct.
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