hello A K M Zakir Hossain so, after getting the knockout, you're not making single cell KnockOut cell line? you're just maintain the whole population (knockout + WT) under antibiotic selection and doxycycline induction?

In that condition, i have several point of view:

1. Your system maybe get a epigenetic silencing, maybe on one of the promoter. Check the mRNA expression of each component.

2. Treating the cell with continous antibiotic by the time can make the cell become resistant, even the wild type one. So there is a probability, after several passage, the Wildtype population is overcoming your KO cell population.

3. issue number 2 also can happen because Cas9 induction actually is a toxic condition for the cell, so it can make the cell unhealthy and more susceptible to dead.

Also,you mention about "showed no reduction in protein expression". If you want the KO cell, you should be getting no protein at all, if you just want to reduce the protein expression, youre doing KnockDown, not KnockOut.

Anytime you use a lentiviral transfection system on transformed "lab rat" cells, you ideally need to use them within 3 to 5 passages or they'll literally "kick out" the plasmid and you will lose your knockout or fluorescent readout system. I used a Lipofectamine system and this happened to me often.

This happened to me with HUVEC, HEK293T and HeLa cells while primary cells extracted from mice tended to be more stable.

Also, using old cells to transfect is a bad idea. If you ordered the PANC-1 cells from ATCC or got them from someone else, you need to know what passage they are when you start them. Most transformed cells are most receptive to stable transfection with lentivirus at around P1 to P10 or P11. If you're at P12 right out of the liquid nitrogen and you passage twice to expand your cell counts...then your transfection might only be good for 1 or passages. Adding extra transfection reagent doesn't really help either since the transfection isn't the issue, the cells kicking it out seems to be.

So, ideally:

1. Use your PANC-1 cells at early passages and never to 100% confluence

2. The transfection will be pH sensitive whether the literature says so or not. FBS also interferes with transfections a lot of times as the serum has unknown things that tend to soak up the virus and reduce efficiency

3. Always have fresh medium and, after the transfection, leave the cells undisturbed (mechanically) as the cells I worked with seemed to be less able to adhere for a little while (few hours) after transfection

4. Passage once to expand then plan to use all the cells in the next passage.

5. In my experience, liquid nitrogen also reduced the efficiency of my readout so I would never freeze away transfected cells, even if the company rep said it had no effect

Lentivirus and CAS9 don't always work well together, especially if the CAS9 PAMs aren't close enough to the target DNA...I've had several instances where we got the CRISPR/CAS9 system to work the first couple times then never after that. PAMs are short and can be point mutated or otherwise changed and inactivated apparently.

What you are doing can be very frustrating and getting a stable system will require months of work in some cases. We've all been there. Good luck!!!

Bryan J. Mathis Thank you very much, sensei, for sharing your valuable experience and detailed explanation — it really helps clarify the challenges I’m facing. I now understand better how passage number and cellular adaptation can impact lentiviral stability.

In my case, I did observe successful knockout at the initial stage after transduction — my target protein was completely absent in the bulk population, as confirmed by Western blot. Encouraged by that, I proceeded to single-cell cloning under antibiotic selection, but unfortunately, none of the clones retained the knockout.

Even more puzzling, I’m now no longer able to detect any KO even in the bulk population, despite following the same DOX induction protocol. This aligns with what you mentioned — the cells may be silencing or eliminating the vector over passages.

Your point about not freezing transfected cells and using early-passage cells is very insightful. I may need to restart from early passage parental cells and carefully time the transduction and downstream experiments within a narrow passage window.

Thanks again for taking the time to share your perspective — it's truly helpful and motivating.

Leonardo Tejo Gunawan Thank you so much for your detailed feedback and insightful points — I really appreciate your time and perspective.

Yes, at the initial stage after transduction, I observed complete knockout of my target protein in the bulk cell population, confirmed by Western blot. Encouraged by that, I attempted to isolate single-cell clones using antibiotic selection, but unfortunately, none of the clones retained the knockout. Later, even the bulk population lost the knockout phenotype, despite continuous doxycycline induction and selection pressure.

Your suggestions really resonate:

Regarding your final point — I completely agree: if there's still protein expression, it's not a complete knockout. Initially, I had true KO (no protein band), but over time, the phenotype was lost — likely due to the factors you mentioned.

I now plan to restart from early-passage parental cells, carefully monitor expression levels, and consider minimizing selective pressure duration to avoid resistance.

Thanks again for your valuable insights!

A K M Zakir Hossain , this phenomenon is common in actual operation. Possible reasons include:

The gene is silenced. After the lentiviral vector is integrated into the genome, especially in systems containing inducible promoters, the promoter may be epigenetically silenced during long-term culture, resulting in reduced expression of Cas9 or sgRNA, affecting the knockout efficiency.

Insufficient antibiotic selection pressure or the emergence of drug-resistant cells.

It is recommended to re-perform monoclonal screening, optimize antibiotic screening and induction conditions, and detect expression levels. In particular, single-cell cloning and expansion after DOX induction should be performed to screen clones with high knockout efficiency to avoid the influence of population heterogeneity.

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

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

Lucian Miles, Thank you very much for your valuable comment. I appreciate your suggestion.