Need help: LentiSAM/LentiMPH system for Western Blotting after lipofectamine transfection

We are currently establishing LentiSAMv2 and LentiMPHv2 system in our lab. Our first aim is to show the system works in NIH3t3 cells using lipofectamine transfections (control-guideRNA or gene-specific guideRNAs cloned into LentiSAMv2 backbones and co-transfected with LentiMPHv2 (1:1)) and lysis after 5 days incubation for western blotting. However so far, we were not able to see a robust overexpression of the targeted genes using different guideRNA templates extracted from the online tool :/ Has anyone worked with such transient transfections of LentiSAM plasmids in NIH3T3 cells?

We are not using any selection method in NIH3t3 cells after transfection(such as Blasticidin treatment, due to the marker on the LentiSAM) since we would like to transfer this protocol into neurons (using the available Cre-dependent SPH (SunTag-p65-HSF1) mice from Jacksons or generating lentiviruses directly from these constructs)in which we cannot generate clonal lines thru selection. Could lack of antibiotic selection be the problem? Since we often get high transfection rates and effects when we transfect classical shRNAs or expression constructs using lipofectamine (in 2days without any antibiotic selection), I didnt think this is the problem. But maybe LentiSAM/LentiMPH method needs more incubation time to see effects in protein levels or something else that we cannot think of?

** I recently saw the paper "Chavez, Alejandro, et al. "Comparison of Cas9 activators in multiple species." Nature methods(2016)" in which they transfected the LentiSAM system from Konermann 2014 paper to NIH3t3 cells with lipofectamine and did qPCR after 2 days. their transfection ratio was 25ng:10ng:100ng for dCas9:guideRNA:SAM factors (which is different from mine), but again they did not do any westernblots.


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Fuchsia Kelpiealmost 4 years ago

For troubleshooting, I would recommend doing RT-qPCR to see if there is any transcriptional upregulation. We have a good protocol for doing this easily in the Joung Nat Protocols 2017 paper.

I have never work with 3T3 cells, but I have had experiences where Cas9 SAM robustly upregulated the mRNA of the gene of interest, but not the protein, due to post-transcriptional silencing.

Sienna Pixieabout 2 years ago

Following this topic, how often did you observe post-transcriptional silencing? Is it associated to the SAM overexpressing system or we should expect the same process if we overexpress the same target gene exogenously with a conventional method such as a expression plasmid?

Fuchsia Kelpieabout 2 years ago

I have seen this for 2 cases, and this seems to depend on protein and cell type. The protein expression will be silenced via the UTRs that are inherently present when you upregulate the gene at the endogenous locus, such as through SAM CRISPRa. If you are seeing this, then you can avoid it using cDNA overexpression (ie, the ORF without the UTRs).

Sienna Pixieabout 2 years ago

Thank you very much!

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