We are creating transgenic zebrafish lines with the TOL2-kit of Dr. Koichi Kawakami. We are trying to incorporate into the genome a B-actin2-humangene-IRES-GFP construct of about 8-9kb. Is this construct too big?
What are the concentration you are using of transposase RNA (is protein a better option?) and vector DNA (M).
Vinothkumar Rajan
I have integrated bigger constructs into the genome using tol2. I typically use 35 ng/ul of transposase mRNA and 25 ng/ul of vector. Sometimes, I have increased the concentration of transposase mRNA until 50ng/ul but with increased mortality of embryos.
Also, seeing at your construct there has always been inconsistent results with IRES in zebrafish and we have now completely moved to P2A that works much better.
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