Advanced RNAi Applications — Difficult Cell Types FAQs

How do I know if my cell type can be transduced?
Which cell lines or types have been tested?
Are MISSION^® shRNA viral particles suitable for transduction of neurons? Does direct infusion of positive control viral solution into the mouse brain result in stable GFP expression?
I would like to know whether anyone has used shRNA on primary human monocytes and if so is there any advice?
Which packaging cell line should I use to make lentivirus?

How do I know if my cell type can be transduced?

The lentiviral particles are pseudotyped with VSV-G Protein. This allows transduction of a wide range of cell types. We have listed cell types that have been successfully transduced with the pLKO.1-puro lentiviral constructs.

Which cell lines or types have been tested?

We have a list of cell types that have been successfully transduced by pLKO.1-puro based lentiviral particles.

Are MISSION^® shRNA viral particles suitable for transduction of neurons? Does direct infusion of positive control viral solution into the mouse brain result in stable GFP expression?

Yes, multiple researchers have demonstrated that MISSION^® shRNA lentiviral particles can transduce neuronal cells, both in culture and in vivo. That said, expression of GFP in some species has proven difficult when driven from a CMV promoter.

I would like to know whether anyone has used shRNA on primary human monocytes and if so is there any advice?

Here is a reference describing how lentiviral shRNA has been used to stably transduce monocytic cells: Unwalla, H.J. et al. Negative feedback inhibition of HIV-1 by TAT-inducible expression of siRNA. Nat. Biotechnol. Dec, 2004 22(12), 1573-8. Epub 2004 Nov 28.¹

We suggest optimizing your experimental conditions with the TurboGFP Control Virus (Product No. SHC003V). You can easily see the transduction efficiency obtained with different lentiviral MOIs in your cells.

Which packaging cell line should I use to make lentivirus?

Most cell lines are NOT ideally suited to produce lentivirus with the MISSION^® shRNA library. We suggest using an HEK-293T cell line for generating virus particles. In addition, you'll need to use packaging vectors derived from HIV. We recommend using our Packaging Mix (Product No. SHP001) for optimal high titer virus production.

Contact Us

For questions about the library, pricing and quotes or other concerns, please e-mail us at: MISSIONRNAi@sial.com.

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References

1. Unwalla HJ, Li M, Kim JD, Li HT, Ehsani A, Alluin J, Rossi JJ. 2004. Negative feedback inhibition of HIV-1 by TAT-inducible expression of siRNA. Nat Biotechnol. 22(12):1573-1578. https://doi.org/10.1038/nbt1040