I have been trying to transfect primary mouse neural progenitor cells (wildtype and heterozygous for our gene of interest) with a plasmid expressing dCas9-VP64 and a plasmid expressing sgRNA for CRISPR activation of the gene.
We have tried a MSCV plasmid expressing dCas9-VP64 under the MSCV-LTR promoter, and we successfully activated the gene's expression. However, our goal is to observe the rescue of phenotypes in neurons, and retrovirus cannot infect non-dividing cells. So, I tried cloning the dCas9 onto an AAV backbone plasmid and it is expressed under CMV and T7 promoters. We used the same sgRNA plasmids. But this led to unsuccessful activation.
Can someone please provide a possible explanation for why I see these varying results after transfection (nucleofection) between these plasmids? Is the cell type the limiting factor? Is LTR just that much better for expressing genes in proliferating cell types compared to CMV?
Dear Prerona,
The strength of the different promoter can of course influence the gene expression but I'd rather see your concern from another angle:
I would like to offer you the possibility to test our NeuroMag transfection reagent as an alternative, in order to increase the transfection of you neural cells with plasmid DNA without employing viral vectors.
To be brief, NeuroMag was originally developed to transfect specifically primary hippocampal and cortical neurons it has been successfully demonstrated since almost two decades now, to be highly efficient on almost every type of primary neurons (cortical, hippocampal, motor neurons…) from several species (rat, mouse…), on also “neuronal cell types”, such as astrocytes, oligodendrocytes, glial cells and what is of interest here, neural stem cells.
Moreover, NeuroMag can deliver any kind of nucleic acids (DNA, mRNA, ODN, siRNA…).
you can refer to the following publications :
Would you like to give NeuroMag a try, please do not hesitate to contact me via ResearchGate or directly at: [email protected]
best regards,
Cedric
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