Are there any suggestions on how I can enhance the probability to only integrate one copy of my tol2 flanked DNA fragment into the zebrafish genome? Does adding meganuclease help? I always end up having multiple insertions.
Vanessa Barone
Hi Barbara,
one cannot control the number of copies inserted with tol2, and I believe the same is true for meganuclease driven transgenesis. Multiple insertions are the norm with these methods. What you can do is outcross your transgenics to WT as long as you have animals with only one insertion, but this can take several generations. If you really need single copy insertion, I would suggest a CRISPR based approach.
Good luck!
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