Neuronal Expression and pRF4

Hi all,

I’m new to the forums and have just started working with C. elegans again after a long time. I have been trying to express GFP or synuclein in serotonergic neurons or dopaminergic neurons. I created the following plasmids using the invitrogen gateway system

cat-2 promoter - GFP - cat-2 3’utr
cat-2 promoter - Synuclein - cat-2 3’utr
tph-1 promoter - GFP - tph-1 3’utr
tph-1 promoter - Synuclein - tph-1 3’utr

Promoter region is 1.8 kb up to (but not including) the start codon of the ORF. The ORF is the full cDNA of either synuclein or GFP. The 3’ utr is 500 bp in length from (but not including) the stop codon of the ORF. I used the pCG150 plasmid as the expression vector and bombarded DP38 worms according to established protocols. Screening the worms 2-3 weeks later I was able to isolate dauers - suggesting that the unc-rescue fragment in the expression vector is working.

However, I am not seeing any GFP expression in these worms. Any suggestions?

I am also planning a second approach to this by sub-cloning the cat-2/tph-1 promoter in to HindIII-BshTI sites of the MCSI of the pPD95_75-mCherry plasmid and microinjecting the worms along with the pRF4 marker. Then expressing synuclein by removing the mCherry fragment using KpnI and EcoRI and sub-cloning in the cDNA of the gene I want. Does this seem like a reasonable approach that would work?

To do this I need the pRF4 plasmid, which I don’t have. Does anyone know where I can obtain the pRF4 plasmid? I have searched the web but haven’t found anything.

Thanks for reading and any help you can give me.