Recently I tried to use my nanoparticles carrying GFP mRNA to transfect liver cells in vivo. And my plan is to figure out which kinds of liver cells (Like hepatocytes, Kupffer cells...) can be transfected by GFP mRNA using flow cytometry. And I injected 50 ug GFP mRNA for each mice (I tried 6 ug Luc mRNA for each mice before and the signals is pretty high in liver which shows my formulation looks good). But there is no GFP+ cells detected compared to PBS group after testing liver single cells.
Here is what I thought, GFP maybe not a good choice because of autofluorescence. Maybe I can used mCherry mRNA instead (Although Ai9 report mice maybe the best choice, it is hard to get one here.)
Another solution is I use a GFP-antibody and used another fluorescent labelled secondary antibody to improve the signals.
Do you guys have some suggestions? Thanks in advance!
Dear Yang, you may use another method to detect GFP fluorescence from tissue samples. We used a GFP quantification kit. Check our article for the protocol:
https://www.researchgate.net/publication/326083470_Glomerulosclerosis_in_transgenic_rabbits_with_ubiquitous_Venus_protein_expression
Nándor Lipták Dear Nándor thanks for your kind help. Because we want to quantify the GFP transfection efficacy in specific cell groups of liver (like hepatocytes , Kupffer cells) instead of the whole tissue. Maybe GFP quantification kit may be not works here. But anyway, thanks for your kind help.
@Bowei Yang Good point. That GFP quantification kit may help you to explore if there is any GFP signal in the liver after the transfection. That information could be useful to identify what the actual problem is with the transfection.
We have used LipExoGen’s EGFP mRNA LNPs and they worked good for us. I am wondering if your mrna been degraded?
Yuanyi Wei Thanks Yuanyi for your reply! I finally solve this problem. I found out GFP-mRNA translate later than Luc mRNA. So when I delayed my detection time and I finally can detect some GFP+ cells.
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