hello Shahd Abu Kamel , you transfect a plasmid for transient expression in mamallian cel right? or you transfect with plasmid containing your Gene interest (including Puro and GFP) and containing mamallian-suitable ORI, like EBV or SV40 ORI?

if your plasmid doesnt contain these mamallian-suitable ORI, when your cell proliferate or you make them freeze, the plasmid you succesfully transfect to 95% cell before will be released by the time. In example, in HEK293cell, plasmid maybe only retained in the cell around 4-7 days, it maybe longer if you keep treat with antibiotic, puromycin in example, but at some point, it will be released. For note, even your plasmid contain EBV or SV40 ORI, it will just make the release plasmid longer, but not stay forever in the proliferating cell.

Thank you for your response Leonardo Tejo Gunawan , I transfected the cells using lentiviral system to generate a stable cell line . My gene of interest and GFP are under the control of a minimal CMV promoter, while puromycin selection is driven by the Ubc promoter.Since I used lentivirus to transduce my cells, I expected all of them to have the plasmid. However, I'm not sure why that’s not the case.

hello Shahd Abu Kamel . Wait a minute, i'm bit confused.

First. in the earlier question, you tell transfection of plasmid containing TetOn system, but now using minimal CMV promoter. Could you please detailing about the plasmid map? In example like this CMVProm-GOI-T2A-UbcProm-Puro etc.

Second, what cell line you used?

Third, you clarified that you transduced the cell with lentivirus so you hope all the cell line will have plasmid. Lentivirus will not make the cell containing plasmid. Lentivirus only transfering the genetic material that you inserted before in the plasmid.

So in simple, heres the workflow.

1. You produce lentivirus in cell (i.e. 293T) using 3 plasmid containing vsvg (envelope), pol-gag (packaging), and plasmid containing your GOI cassette flanked by 5' and 3' LTR. In several days, the cell will produce lentivirus containing your GOI cassette and you will harvest it.

2. you used that lentivirus (no plasmid anymore) to transduce your target cell line to make stable cell line.

and then let's assume your situation (correct me if im wrong)

1. Made a stable cell line via lentiviral transduction.

2. The construct includes: Tet-on/off system (for what gene?) GFP and gene-of-interest (GOI) linked by P2A using Minimal CMV promoter for GOI + GFP Ubc promoter for puromycin resistance

3. Selected with puromycin and cryopreserved.

4. Now, → only ~56.8% of cells express GFP.

So in this case the issue is not about plasmid loss (as in transient transfection), but rather transgene silencing or heterogeneity in integration/activation.

These are maybe on of your problem:

• Heterogeneous integration: Not all cells may have integrated the lentivirus equally (multiple copies vs. one copy vs. none). Even after selection, low MOI transduction can result in mosaic populations.
• Silencing of the minimal CMV promoter: Minimal promoters can be prone to silencing over time, especially in certain cell types. The Tet-on system relies on a strong and consistent rtTA expression—any variability in that can cause partial induction.
• Suboptimal Dox induction or loss of rtTA expression: If the Tet transactivator (rtTA) isn't stably expressed or is silenced, even with Dox, you won’t get full induction.

Hi Leonardo Tejo Gunawan you are correct. The plasmid map as following , tet operator-minimal CMV promotor-GOI- eGFP p2a-Ubc promotor-rtTA3-puro. I am working with AGS cell line . I’d like to determine whether the observed reduction in GFP expression is due to silencing of the minimal CMV promoter or loss of rtTA expression. What strategies would you recommend to identify the root cause and to overcome the issue?

thank you

Shahd Abu Kamel , there are several possible reasons for your problem:

Some cells lost plasmids or integrated fragments: During freezing, thawing and amplification, puromycin was not added continuously, and some cells may have lost fragments with resistance genes and Tet system, resulting in only some cells expressing GFP after induction.

Epigenetic silencing: The promoter of Tet system may be epigenetically silenced after a long period of non-induction or non-screening, resulting in some cells no longer responding to doxycycline.

Increased cell heterogeneity: After freezing and amplification, the homogeneity of the cell population decreased, and some cell expression systems were silenced or lost.

It is recommended to re-perform puromycin screening before doxycycline induction. The mainstream operation recommends that after the cells are frozen and thawed, antibiotics (such as puromycin) should be added after the cells resume growth to remove cells that have lost resistance genes and ensure the stability and homogeneity of the population.

Or you can refer to the following document for more tips on Doxycycline-induced modeling:

Method Doxycycline-Induced ON-OFF (Overexpression-Knockout) System ...

Thank you Lucian Miles ,for your reply . I selected the cells with puromycin for 48 hours after thawing, before inducing them with DOX, but it didn’t work. So, I repeated the infection, and this time I got 95% GFP-positive cells based on the FACS results. I tested other gastric cancer cell lines besides AGS and was able to detect upregulation by western blot. However, in AGS cells, I’m still unable to upregulate the gene, even though it had worked in the past.

Shahd Abu Kamel , you did try many methods to solve the problem.

I think a big reason why the experimental results did not meet expectations is the particularity of AGS cells: the activity of the Tet-on/off system in AGS is abnormal.

The Tet-on/off system depends on the expression of rtTA. So it is recommended that you detect the expression of rtTA in AGS cells by WB or qPCR, and compare the expression levels in other successful cell lines to check whether there are expression differences. If the expression of this gene in AGS cells is indeed very low, it is recommended to additionally transfer the rtTA expression plasmid into AGS cells to enhance the activity of the Tet-on/off system.

One point worth noting is that you screened GFP-positive cells by re-adding antibiotics, but the expression of the target gene in AGS is still very low. This means that even if GFP is expressed normally (plasmid integration and promoter initiation are normal), the target protein may be rapidly degraded due to the specific environment of AGS cells (such as high expression of proteases, folding barriers), resulting in the inability to detect "upregulation" by WB.

You also need to pay attention to whether the effective concentration or action time of Doxycycline in AGS is insufficient.

Different cell lines have different uptake and sensitivity to Doxycycline. The 2.5 μg/mL (5.62 μM) you used may be too low for AGS, or DOX is metabolized faster in AGS. You can try a higher concentration of Doxycycline (reference PMID: 31944316) (e.g. 5 μg/mL, or 10 μM), or shorten the dosing interval (e.g., every 12 hours) to ensure continuous induction of stress.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

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