We would like to clone two different proteins, one tagged with GFP and the other with RFP into the same attB plasmid and integrate them at the same site in the flies. I imagine that after the stop codon of the first trasgene, adding a spacer and the promoter again followed by the second transgene should work. Does anyone have experience with this? Is there anything else to be considered? What would be the optimal spacer length between the two transgenes?
Thank you!
Hello Sreemukta.
We've done a similar tagging works with GFP-LacZ fusion gene constructs under unbiquitous pCMV and neurospecific pTa1 promoters. If you give a spacer between the two reporter gene without promoter from the second reporter, it would make such desired constructs. and reliable expressions in the same spots.
- Isolation of neuronal precursors from differentiating P19 embryonal carcinoma cells by neuronal Tα1-promoter-driven GFP (2001)
- Dual reporter genes enabling cell tracing with viable and reliable selection of various cell types (2006)
Regards.
Hello Sreemukta,
yes one can express both proteins from the same construct. I created a construct for site-directed integration having one gene under control of the HuC (elavl3) promoter and another one under the alphaA-crystalline promoter. Both expressed nicely in zebrafish. However, if you have large genes or promoters be aware that handling big plasmids can be tricky.
Best regards
Daniel
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