Packaging LentiCRISPR

[Justin G]

Hello,

I've cloned some targeting sequences into the LentiCRISPR vector and am about to try producing virus to transduce my target cells.  I was reading over the protocol available for doing so and I saw that it recommended using pVSVg and psPAX2 available from Addgene.  We typically use the Invitrogen "Virapower" packaging mix in our lab for producing lentivirus.  The Virapower mix consists of 3 plasmids:

-pLP1 (containing CMV-driven gag/pol) 

-pLP2 (containing RSV-driven REV) 

-pLP-VSVG (containing CMV-driven VSV-G).

I was hoping to get going with packaging rather than purchase a new plasmid from Addgene and wait for our legal department to approve the MTA, then wait for it to arrive, wait to grow it up, etc.  Does anyone have experience using the Virapower system with LentiCRISPR, or know if it should be compatible (or how I can determine that)?

Thank you in advance for any help you could provide.

-Justin

[Neville Sanjana]

Hi Justin,

I haven't used it but the Virapower packaging mix sounds like a 3rd generation lentiviral system. This should be compatible with lentiCRISPR. You should validate your viral production by making sure (with, say, a small scale transduction) that you get puro resistant cells. This will prove that functional virus has been made.

Hope that helps,

- Neville

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[Justin G]

Thanks.  I will try that, and report back here how it worked.

-J

[Sapna Shah]

In relation to this topic -

We've been using psPAX2 and pMD2.G (http://www.addgene.org/12259/) to package lentivirus. Can I use pMD2.G to package lentiCRISPR or do I have to use pVSVg?

Thanks,
Sapna

[Neville Sanjana]

pMG2.G is fine to substitute for pVSVg. The only difference between these plasmids is CAG (in pMD2.G) vs CMV (in pVSVg) promoter. Either works great for making lentiCRISPR.

Best,

- Neville

[Giovanni Pacchiana]

hi there! I'setting up all the stuff to use the lentiCrispr system... In our lab we usually use the pIlVV01 (rev), the pMD2.vsvg and the pMDLg/pRRE packaging plasmids...do you think are ok?

[Neville Sanjana]

Hi Giovanni,

I'm not familiar with that particular Rev plasmid but that should work fine. See here for details on using 3rd generation packaging vectors: http://www.addgene.org/12251/

best,

- Neville