Has anyone here tried to make PCR fusion GFP construct to express your gene of interest under a heterologous promoter? I am planning to do this for my gene of interest. Any insight/ suggestion would be helpful?I am thinking of pooling three different PCRs (promoter+ gene+GFP) to make the “construct” for direct injection
We’ve had good luck with the NEB Gibson Assembly kit. http://www.neb.com/nebecomm/products/productE2611.asp
So, with the NEB Gibson kit, did you use the same primer designing technique that involves overlap of one fragment with that of the next?
To answer in part your original question, yes, we have done a similar type of experiment, to generate tissue-specific expression constructs for fasn-1, a gene that spans more than 10kb, by directly injecting 4 overlapping PCR products. The last 3 were common to all constructs, and the first one used either the endogenous promoter or the epidermal col-12 promoter, the intestinal mtl-2 promoter, or the neuronal rab-3 promoter. See http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3073241/figure/F2/. You can find details in the article by Lee et al. in Virulence at http://www.landesbioscience.com/journals/virulence/article/10974/. There’s no reason to think that it wouldn’t work with a 3’ GFP gene fragment.
Hope this helps
Thank you so much for the link to your paper, I was wondering how much overlap did you have between the tissue specific promoter and your gene PCR fragment , I didnt see the detail in the paper (might have missed it)?
It’s in the methods section: “Each PCR fragment overlapped its neighbor by at least 900 bp”.