I'm using a transposon tol2 vector (pT2AL200R150G - 10.1534/genetics.106.060244) to create transgenic zebrafish line.
We first inserted our gene of interest (GOI) into the BamHI site of the vector, which was fused with the eGFP sequence already present in the plasmid. Next, we replaced the native promoter of the vector (ef1a) with another promoter by digesting the vector with XhoI and SalI and ligating the insert via in-fusion ligation.
After cloning and validating, we injected the vector along with the transposase mRNA into the one-cell stage zebrafish embryo. The following day, we observed GFP expression, indicating expression of the GOI fused to it, mainly in the vector with native constitutive promoter Ef1a+GOI. However, after a few days (3/4 days), the fluorescence expression gradually disappeared until it was no longer visible. This is unexpected, as we injected transposase RNA, which should theoretically insert the transposon into the animal's genome. We are wondering if this is a normal occurrence and whether crossing F0 will result in expression in the offspring.
P.S. We performed PCR on the injected animals for both GOI and GFP, and although we observed amplification of the sequences, we did not detect any expression.
The transient expression of your transgene in zebrafish embryos followed by a loss of fluorescence could be due to several factors. While the Tol2 transposon system is commonly used for stable integration of transgenes into the zebrafish genome, transient expression is not uncommon, especially in the absence of selection pressure. Here are some potential explanations for your observations:
Regarding your plan to cross F0 fish to establish a stable transgenic line, it's possible that stable transgenic lines could still be established even if F0 embryos show only transient expression. However, the success of this approach may depend on the factors mentioned above.
To troubleshoot and improve your transgenic strategy, consider the following:
- Optimize Promoter Choice: Ensure that the promoter you have chosen is suitable for driving robust and stable expression in the tissues of interest.
- Increase Transposase mRNA Concentration: If integration efficiency is an issue, consider optimizing the concentration of transposase mRNA during injections.
- Use Positive Selection: Co-injecting a Tol2 transposon carrying a selectable marker (e.g., antibiotic resistance gene) along with your gene of interest can help select for embryos that have successfully integrated the transgene.
- Test Different Promoters: If the replacement of the Ef1a promoter is affecting stability, consider testing different promoters that are known to drive strong and stable expression in zebrafish.
- Consider Tissue-Specific Promoters: If your gene of interest is meant to be expressed in specific tissues, consider using tissue-specific promoters to drive expression in those tissues.
Performing additional experiments, including crossing F0 fish to assess germline transmission and stable expression in F1 generations, can provide valuable information about the success of your transgenic approach. If the issue persists, consulting with experienced researchers in zebrafish transgenesis or molecular biology may provide additional insights into optimizing your experimental design.
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