In primary cells how long should I wait after transfection before I can assay for gene expression?


For delivery in primary cells or any cell model, gene expression is dependent on the downstream assay required for the experiment. A typical window or range is anywhere from as early as 12 hours for expression from mRNA delivery, 24-48 hours for evaluation of expression of a fluorescent protein, to 72-96 hours for evaluation of genome editing tools like TALs or CRISPR. However, there are factors that could influence the efficiency of expression such as size of the DNA or mRNA, the promoter for the DNA, age and health of the cells, the half-life of the protein being expressed, etc..

On another note, a little tip we can offer, if you are trying to improve delivery in primary cells, would be to try an mRNA version of your gene of interest. The reasoning is that mRNA only requires entry to the cytoplasm of the cell as opposed to the nuclear localization that is required for DNA; and since primary cells tend to be in a post-mitotic state, with minimal nuclear envelope disruption, delivery to the nucleus has proven to be quite challenging. Life Technologies offers multiple in vitro transcription kits that are complete with the necessary items to make mRNA in the lab; an ARCA cap, the nucleotides and a RNA specific polymerase are included in the kit. The only requirement is a linearized vector or PCR product of the gene of interest containing a T7 or SP6 promoter. More information can be found on the Life Technologies website.

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