PCR fusion constructs

Model Organism: Worms Methods & Reagents
1

Hi All

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

Thanks :slight_smile:

2

There is a wormbook chapter dedicated to this matter

http://www.wormbook.org/chapters/www_reportergenefusions/reportergenefusions.html

good luck

A

3

We’ve had good luck with the NEB Gibson Assembly kit. http://www.neb.com/nebecomm/products/productE2611.asp

4

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?
Thanks

5

Hello

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

Jonathan

6

Hi Jonathan

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)?

7

Hello

It’s in the methods section: “Each PCR fragment overlapped its neighbor by at least 900 bp”.

Good luck