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I need to express a selection marker on a plasmid in human cells (e.g. HEK293). However, I am using a cytoplasmic vector derived from a non-human virus with its own mechanism for transcriptional initiation. This means that the expression level of any protein marker will suck: low copy number, very weak promoter, unstable transcripts - these are things that I cannot change in this precise biological context.

Most selection markers (for instance KanMX) come with their own black-box promoter and so I do not know whether they rely on a high number of proteins being produced.

What would be a marker most likely to be selectable despite a very low expression level? I could not find any resource comparing the protein copy numbers of classic markers.

I need to be sure that the cells that cannot survive the selective pressure died because they did not have the marker, and not because the marker was too weakly expressed to protect cells that have the marker. The standard approach would be to use a regular antibiotic selection and just work with lower concentrations of antibiotics but the number of false positives would most likely be too high.

I can manipulate the host cell to some extent, so I can use antibiotic selection but I can also complement a conditional deletion of an essential gene (auxotrophic marker or non-metabolic genes).

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  • $\begingroup$ Are you trying to select for the plasmid or for expression of a gene from that plasmid (e.g. by a translational fusion of the gene with a marker)? Can you provide more information about the "technical constraints"? $\endgroup$
    – tyersome
    Commented Jan 22, 2020 at 21:13
  • $\begingroup$ @tyersome I would be just selecting for the presence of the plasmid after N divisions. Ultimately I would express other things (e.g. luciferase) from the same plasmid but before optimizing that aspect I need to know that the plasmid can stay there for a while. By technical constraints I mean that the plasmid comes with a pre-defined origin of replication and mechanism of transcription initiation, but is derived from a non-human mammalian virus, so it will most likely not "work" as well as in the original host (in which expression levels are already low) $\endgroup$
    – Mowgli
    Commented Jan 22, 2020 at 21:47
  • $\begingroup$ @Mowgli If your plasmid does not have a strong promoter then how are you planning to use it for your later experiments with luciferase etc? Regarding whether the plasmid will stay: for how long would you be doing your experiment (HEK293 divides slowly)? $\endgroup$
    – WYSIWYG
    Commented Jan 24, 2020 at 10:25
  • $\begingroup$ @WYSYWIG designing and testing better promoters on this platform is part of the ultimate goals. I expect luminescence (or other techniques based on signal integration) to be sensitive enough to detect even the very small expression level of the original construct. I am expecting the plasmid to be maintained for over a week - in theory it will stay "forever" like a genomically integrated lentiviral vector, if it has a replication mechanism (already built in) and a selection system (this question) $\endgroup$
    – Mowgli
    Commented Jan 24, 2020 at 13:33
  • $\begingroup$ Based on your reply it doesn't seem clear why you can't use a standard promoter to drive your selectable marker. Why do you feel constrained to use a "very weak promoter"? Why do you think the transcripts for the marker will be unstable? $\endgroup$
    – tyersome
    Commented Jan 25, 2020 at 21:52

1 Answer 1

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Flow cytometry looking for your plasmid.

These folks labeled their plasmid with a proprietary fluorescent marker, then used flow cytometry to detect cells which had taken up the plasmid.

A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells

An alternative and more direct method to using fluorescent reporter genes is to directly label nucleic acids with fluorescent dyes to track their intracellular delivery [16]... Using the non-enzymatic Label IT® Tracker TM Kits, any plasmid can be custom labeled in a simple one-step chemical reaction before introduction into mammalian cells [18]. Thus, both subcellular localization of the labeled DNA and expression reporter transgene can be monitored simultaneously following introduction of the labeled plasmid into mammalian cells [16, 18]...

Herein, we demonstrate the development of a flow-cytometric assay to determine transfection efficiency by labeling a reporter plasmid with Label IT® TrackerTM. This method does not depend on co-transfection of two different plasmids and simultaneously quantifies cell death, uptake of the labeled plasmid during transient transfection, and expression of the target protein.

Even if only a small fraction of your cells take up your glowy plasmid, you will sort those out from the rest with flow.

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  • $\begingroup$ While the technique is interesting, it does not create a constant selective pressure like an antibiotic cassette or an auxotrophy marker, which means that I would have to sort the cells every few days to maintain a positive population... Additionally, chemical labelling prior to transfection cannot detect replication products. $\endgroup$
    – Mowgli
    Commented Jan 23, 2020 at 0:00

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