I am currently isolating and cultivating primary neurons from E18.5 C57BL/6 mice. I am planning to transfect them with both CRISPRs and a selection vector that will express RFP when the Cas9 is cutting.
To do this I need to both transfect the neurons and FACS sort them for RFP.
Does anyone have a protocol on how to achieve this?
Thankful for any input.
Best,
Linn
I'm not sure you'll find an exact protocol for CRISPR in primary neurons with FACS afterwards, but you should still be able to synthesize different procedures into your overall research. There's some kits for CRISPR in murine neuronal cell lines (see https://altogen.com/product/neuro-2a-transfection-reagent-neuroblastoma-cells-ccl131/ ) but you will still have low transfection efficiency - it's primary cells after all.
I have got best results with nucleofection (check https://bioscience.lonza.com/lonza_bs/CH/en/nucleofector-technology). They have specific kits based on cell types. Plate them back and let them grow for 48 -72 hours after nucleofection before proceeding with FACS.
Hello,
I agree with the previous answers, the format of CRISPR components and the delivery method are very important with difficult cells as primary neurons. You will have a serious limitation by using plasmid vectors except maybe with electroporation.
Have you ever thought about using a mRNA approach or directly RNP?
https://www.researchgate.net/project/VIROMERR-an-innovative-technology-for-transfection/update/5c93786a3843b03424324c64
Best, Sandra
Hello
Kindly look at these links , hope help you
Article Inefficient Type I Interferon-Mediated Antiviral Protection ...
Article Snx14 Regulates Neuronal Excitability, Promotes Synaptic Tra...
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