Does anyone here can provide me some background for generating the transgenic line in zebrafish? I wish to generating GFP specific expression in microglia by inserting EGFP marker in apolipoprotein E locus via BAC, but I have no idea on the genome construction part. Hope someone can help me out. Thanks!
Hello, Alex.
If you want ApoE gene with glow under CMV promoter-ApoE-GFP gene construct for your transgene, you can see all cell types glowing, including microglia of the transgenic line.
Or If you want microglia-specific ApoE-GFP expression, constructing transgene as microglia-specific gene promoter-ApoE-GFP construct, you can see only your glowing microglia, overexpressing ApoE-GFP fusion proteins theoretically.
So, this has to be decided what you want to do.
If your choice is the second, then you have to start to search microglia-specific genes in zebrafish (which I doubt gene promoters sequenced), mouse and human gene promoters from the public genome bank.
You could begin with TMEM119, Iba1, F4/80, CD11b and CD68 genes. Get the promoter sequences to be cloned, use them to fuse to ApoE cDNA-GFP, making a plasmid vector, pIba1-(or pTMEM119-, pF4/80-)ApoE-GFP.
Ref
-ionized calcium binding adaptor molecule 1 (Iba1): Molecular Brain Research 57(1):1-9 · July 1998
-transmembrane protein 119 (TMEM119): www.pnas.org/cgi/doi/10.1073/pnas.1525528113
-F4/80, CD11b, CD68: Gene Therapy (2003) 10, 657–667
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