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Amplify lentivirus


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#1 milla

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Posted 28 June 2010 - 08:54 AM

Hi Guys!!

I got a lentivirus from another lab and now I'd like to amplify it.
I have done maxiprep to amplify plasmid but I have no idea on how I should operate for a lentivirus.

I hope someone can give some suggestions.

Thanks!

#2 laurequillo

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Posted 28 June 2010 - 08:06 PM

[quote name='milla' date='Jun 28 2010, 06:54 PM' post='77326']
Hi Guys!!

I got a lentivirus from another lab and now I'd like to amplify it.
I have done maxiprep to amplify plasmid but I have no idea on how I should operate for a lentivirus.

I hope someone can give some suggestions.

You can use it as a normal plasmid in transfection, or use it to produce virus.
To produce virus you need to cotransfect your construct plus some plasmids that produce the viral proteins gag,pol and env. Normally you neee one plasmid for gag and pol, and another one for the envelope. Then you transfect 293t with the three of them and your cells will produce the virus. You collect them and use them to infect other cell lines.

Here you have an overview:
Lentivirus
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#3 milla

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Posted 29 June 2010 - 01:09 PM

Let me see if I understood correctly...
I use the lentivirus as a normal plasmid for E.coli transfection (ex DH5alpha) to make the stock and then I cotransfect 293 cells with my construct to create the virus that will allow me to infect the cell line of interest?

Thank you very much!

#4 laurequillo

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Posted 29 June 2010 - 09:57 PM

Let me see if I understood correctly...
I use the lentivirus as a normal plasmid for E.coli transfection (ex DH5alpha) to make the stock and then I cotransfect 293 cells with my construct to create the virus that will allow me to infect the cell line of interest?

Thank you very much!


yeah, you transform your bacteria (normally SURE are better for the shrna), and then you can use the plasmid to transfect your cells (it will give you a transient expression), or use your plasmid to produce virus (and you will use the virus to infect cells in a stably way).
To produce viruses you need your plasmid and another two plasmids (normally).

I hope it is clear
"He must be very ignorant for he answers every question he is asked" Voltaire

"This is SPARTA!"

"Im the goddamn batman"




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