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Know good vector backbone for PT67? - Viral transduction of EGFP to epitherial cell line (Aug/11/2008 )

Hi, all

I am trying viral transduction of epitherial cell line to put GFP in it.
To make viral vector, we have PT 67 packaging cell line, but thinking which backbone I should use.

Do you have any suggestion?

I just need GFP, will not insert any additional gene,
epitherial cell that I am using is from human
I need to have at least one antibiotic resistant gene for selection, and that CANNOT be puromycin

Any suggestion/comment helps,



Unfortunately I dont think that your methodology makes any sense. Your packaging cell line contains all the viral genes necessary to make the viral particles. If you want to introduce GFP as well, then you need to incorporate GFP into the packaging process. It must be in a plasmid that contains the sequences necessary for packaging into a the capsid protein complex. The very nature of packaging cell lines is that in engineering them you've made them completely specific for the complete viral particle you're creating. Once you've engineering the cell line it is permanent, and it only makes the one virus that you engineered. It is the permanent version to what you will read immediately below in the next paragraph. If instead of using a permanent packaging cell line you decide to do a de novo viral production it only takes about 3 days worth of work, most of which is waiting for the cells to grow until you have enough to produce virus. You could easily end up w/ 10^9 viral particles by the end of this week if you wanted.

Not knowing which virus you're making makes answering your question very difficult. If it's an alpha virus then I suggest not using a packaging cell line and instead using a viral plasid purchased from invitrogen or some such and engineering GFP into it under the promotion of a IRES. Then linearizing the plasmid, doing invitro transcription and electroporating into a producer cell line.