I am trying to express shRNA to knockdown my target gene using lentiviral constructs.
I found a 3rd generation lentivector from a bio company. But I don't want to buy their packaging kit.
Just want to know whether I can use this system below instead of their packaging kit:
Packaging system: psPAX2 Map: http://www.addgene.org/12260/
Envelop plasmid: pMD2.G Map: http://www.addgene.org/12259/
I had successful experience to use above for 2nd genereation lentivector ( pLKO.1). But not sure whether they are good for 3rd generation, especially the envelop plasmid.
I would say YEs!
A 3rd generation transfer vector can be used with a 2nd generation packaging system, but a 2nd generation transfer vector cannot be used with a 3rd generation packaging system.
https://www.addgene.org/lentiviral/faqs/#23
pMD.G (vsvg) is an efficient envelope for all three generations!
I would say YEs!
A 3rd generation transfer vector can be used with a 2nd generation packaging system, but a 2nd generation transfer vector cannot be used with a 3rd generation packaging system.
https://www.addgene.org/lentiviral/faqs/#23
pMD.G (vsvg) is an efficient envelope for all three generations!
Yes agreed. It works most for most vectors. In fact, since now you will be tranfecting 3 vectors instead of 4 (lenti + 2 packaging vectors instead of lenti + 3 packaging vectors), you'll probably see higher titers as well. In my hands, it's about 3 fold higher when using the second generation packaging mix combined with a 3gen lenti vector.
Agree with all above. The second generation packaging system does package 3rd generation backbones (it will just also provide tat that is surplus to requirements for the tat independent 3rd gen vectors). The benefit being that you only transfect 3 plasmids instead of 4 will probably give you better titres.
In theory, 3rd generation packaging plasmids should not package 2rd gen backbones because they need tat to produce a full length transcript of the viral genome, and this gene has been deleted from 3rd gen packaging systems. Saying that, I know someone who mixed it up by accident and still got vector with pretty good titre..... we never bothered to go back and work out how or why though.
You might find this webinar useful that goes into more detail on lentiviral methods and 2nd vs. 3rd generation details. The Addgene scientists can also help you choose vectors if you call or email them. https://www.addgene.org/lentiviral/protocols-resources/
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