I want to delivery my cell surface protein as mRNA after in vitro transcription. Previously this had worked great for cytosolic proteins, like EGFP with very high transfection efficiency in many cells using Stemfect. However now I switched the ORF to a cell surface protein, I can only seem to get high expression in HEK293 cells and a little success in HeLa. I am using the same optimised 5'UTR and 3'UTR for cytosolic proteins. I think the transfection delivery is fine but after the mRNA escapes from the endosomes there seems to be some bottle neck in translating and trafficking the protein through the secretory pathway in most cell types I have tried. Seeing as I am using optimised UTR regions for protein expression and not RNA localisation. We have tried using the native UTR of the ORF, such as using the full cDNA clone as template for IVT but still there is some problem.
I know nuclear history of RNA molecules is important for correct trafficking but I read some good papers on chimeric T-cell receptor expression using mRNA delivery and their efficiencies seem to be great.
Sometimes membrane proteins can be difficult to detect by WB. Make sure you add fresh reducing agent such as DTT or B-ME to the sample buffer.
Good luck!
I am trying to express placental alkaline phosphatase and using a fast red AP staining kit from Sigma as used for AP staining of PSC colonies.
I thought about confirming expression from total lysate and membrane preps but struggling to find a good antibody.
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