Does anyone know the best puromycin concentration to select non-small cell lung cancer cell lines transformed with lentivirus?

I have been trying to do lentiviral transduction in the HEK293 cell line to overexpress my protein of interest. I encountered some problems with puromycin selection steps:

1. Almost 50% of cells were alive even after 6ug/mL puromycin treatment

2. HEK293 became suspension cells after puromycin treatment.

Some details:

At first, I tried the puromycin concentration (2.5 ug/mL) recommended by the postdoc in my lab. A majority of cells (HEK293) weren't killed after 3 days; in fact, 80% of them survived. The seeding density is 0.2 * 10^6 per well in a 6-well plate. What's even more strange was that HEK293 became suspension cells.

Then I tried to determine the optimal concentration that kills all the cells in 3 days by treating cells with various concentrations of puromycin (0, 1, 2, 3, 4, 5 ug/mL) in a 6-well plate; Cells were seeded at a lower density (0.1 * 10^6). I also thawed a new batch of HEK293 cells due to the suspension cell issue. Similar things occurred again. The cells weren't killed even at the highest concentrations after 3-day of treatment.

The puromycin should be good since everyone in the lab uses the same batch of puromycin as me. Most of my lab mates transduce with lung cancer cell lines instead of HEK293, so there might be differences in puromycin sensitivity across cell lines.

I'm wondering if anyone has similar experience before? Should I try to generate a puromycin kill curve again with more concentrations (0, 1, 2, 3, 4, 5, 6, 7, 8, 9 ug/mL) and treat them for a longer period? Also, is it normal for HEK293 to become suspension cells?

Thanks so much for your time!!

- A clueless first-year PhD student

I am delivering the pSpCas9(BB)-2A-puro plasmid to SH-SY5Y cells via electroporation, and I am trying to come up with a protocol for the puromycin selection. All of the published literature I can find seems to vary quite a bit in terms of puromycin concentration, duration of selection, when to select. And many of the published methods provide very little detail about it.

Should I place the cells in puromycin-containing media immediately after electroporation or should I wait?

How long should the cells stay in the puro media?

Hello everyone,

In a project I was working, we generated a construct containing Puromycin resistant gene. I want to transfect my GBM cells and select the cells with stable expression of the construct using Puromycin. Which concentration should I use and how long should I treat my cells with Puromycin?

Thanks in advance and sorry for the inconvenience,

Mervenur

I'm trying to make a stable HEK293T cell line that expresses GFP only in the presence of a specific unnatural amino acid (UAA). I packaged lentivirus particles containing my expression cassette, transduced cells, and selected for puromycin and blasticidin resistant cells (used two lentiviral constructs). Using 5 µg/mL puromycin and 6 µg/mL blasticidin, non-transduced cells were killed by 48 hours, while transduced cells remained viable. I expanded these cells and passaged 2x before assaying.

To validate my transduction, I set up an experiment in which I treat cells with or without a UAA and monitor GFP expression (which should only occur in the presence of UAA). When I perform this experiment, I observe that almost all cells are GFP-. Even more perplexing is that ~1% of cells are GFP+, but this phenotype is independent of the presence of a UAA (GFP+ cells without the addition of a UAA). I have attached microscopy images illustrating this issue.

- My expression vector was constructed such that the UAA utilization genes are expressed on the same mRNA as the puromycin resistance gene, separated by an IRES.

- When I transfect my GFP/UAA utilization plasmids into HEK293T cells, they work as they should. This issue appears to be transduction-related.

I am currently working with recombinant lentiviral constructs to transduce/infect a mammalian cell line with also Polybrene at a final concentration of 8 micrograms per milliliter of transduction/infection media. After trypsinizing my mammalian cell line and centrifuging, I resuspended the cell pellet into media containing 1x pen/strep which I then aliquoted into T75 flasks containing premixed Polybrene and Lentiviral constructs. Will the presence of antibiotics affect the efficiency at which my lentiviral constructs infect my mammalian cells? Thank you very much for your time and help!

The oncomine database is shutting down by January 17th 2022, in the meantime I couldn't register in the database, I kept getting errors when trying.

Does anybody have any idea how I can use the database before shutting down, and if there are alternatives for mRNA expression databases across tumor and normal tissue.

I have an lenti-transfer vector encoding puromycin resistant gene driven by PGK promoter. I need to select puromycin resistant clones after LV transduction. What is the best way I can select puromycin resistant clones when I am dealing with such a weak promoter?

I am currently doing LV titration with the same LV packaged vector on HEK cells. I know that the protocol I have followed produces lenti viruses are off high titers. However, majority of HEK cells are dying with 10uL of LV.

How can I overcome this problem?

I'm currently trying to knock down my gene of interest by lentiviral transduction. I am screening 4 shRNAs and a scramble control. The shRNA is under a U6 promoter, puromycin resistance casette is under SV40 and the GFP is under CMV. I generate lentivirus in 293T cells with the psPAX2 and pMD2.G plasmids. Following that I perform functional titring by limiting dilution and flow cytometry in 293T cells. I am working with the prostate cell lines LNCaP, 22Rv1, DU145 and PC3 (later want to try MDA PCa 2b). I have generated stable PC3 polyclonal populations in which I saw a modest reduction at the protein level but did not see a reduction in the mRNA. I am more interested in knocking this gene down in LNCaP, 22Rv1 and MDA PCa2b. My latest attempt with LNCaP (MOI=20) looked to have worked well as cells were expressing high levels of GFP (see attached image) and approximately 50% of cells survived selection in 2 ug/mL for 48 h while all my non-transduced control were dead. After 72 h I transferred from a 6 well plate to a t25, however, very few cells stuck down. Cells have continued to die eventhough I reduced the puromycin to 1 ug/mL. The viral transduction itself doesn't seem to be toxic as cells were growing up until selection. I am at a loss as to why the cells are expressing GFP and initially seem to be puromycin resistant. Any suggestions would be greatly appreciated!

I'm currently working on my fourth try of transformation. Every time the plates have A LOT of tiny colonies (photo attached). Every once in a while there is a larger colony, but when I screen them, they don't contain the recombinant plasmid. The negative control has colonies also, but the plate doesn't have the tiny colonies covering it like the other plates.

I've redone the digestion and still get the same results. I checked to make sure the plates contain ampicillin by transforming bacteria with a plasmid resistant to kenomycin. No bacteria grew when I did this. I've tried troubleshooting, but there are a lot of things I can change, and I'm not sure where to start. Is the ampicillin concentration in the plates not high enough (I've been using 500 uL for 500 mL of agar), am I using too much of my ligation reaction (I've been using all 10 uL), am I plating too much (I've been plating about 350 uL)? Has anyone had this issue before?

Also, when you screen your colonies, what else do you run on the gel? I vaguely remember doing cloning in another lab and when we screened our colonies we ran the uncut vector and the cut vector, but I've only been running the negative and positive controls.