Hello,

yes it's common with stable transfection, especially if the eGFP and the antibiotics resistance marker are under two different promoters. Stable transfection relies on plasmid (or fractions of it) being integrated at random sites in the genome of mammalian cells. Applying the antibiotic to the transfected cells enriches for but does not guarantee the presence of the second transgene (eGFP). With lentivirus or other integration systems it would have been different but what you are observing is pretty normal for stable plasmid transfection.

You may expand your transfected cells in presence of your antibiotics, and then enrich the ones which are eGFP using FAC-sorting. Sort single eGFP+ cells and grow them in presence of the antibiotic, expand them and analyse the eGFP expression. Anything which is still resisting to the antibiotic and expressing the eGFP after 2-3 weeks post transfection will be a bona fide stable integration.

It's best to have a certain number of such clones as, on the long term, exogenous promoters might be silenced. This is unlikely to happen in all the clones that's why it's best to keep a good number of them as backup.

Hope it helps

Francesco

Hello,

yes it's common with stable transfection, especially if the eGFP and the antibiotics resistance marker are under two different promoters. Stable transfection relies on plasmid (or fractions of it) being integrated at random sites in the genome of mammalian cells. Applying the antibiotic to the transfected cells enriches for but does not guarantee the presence of the second transgene (eGFP). With lentivirus or other integration systems it would have been different but what you are observing is pretty normal for stable plasmid transfection.

You may expand your transfected cells in presence of your antibiotics, and then enrich the ones which are eGFP using FAC-sorting. Sort single eGFP+ cells and grow them in presence of the antibiotic, expand them and analyse the eGFP expression. Anything which is still resisting to the antibiotic and expressing the eGFP after 2-3 weeks post transfection will be a bona fide stable integration.

It's best to have a certain number of such clones as, on the long term, exogenous promoters might be silenced. This is unlikely to happen in all the clones that's why it's best to keep a good number of them as backup.

Hope it helps

Francesco

Hi, thanks for the comment.. but my plasmid is a single promoter plasmid . I guess could it be eGFP gene is being slicenced but the antibiotics resistance gene is still being expressed?

I think you misunderstanding Francesco answer. If you look at the map of your plasmid. Most likely, the antibiotic is driven by one promoter while the eGFP is driven by a different promoter (unless both eGFP and antibiotic are driven by the same promoter as a fusion protein. This is very unlikely.).

Also, eGFP signal is strong when it was first transfected. Remember there a multiples copies of plasmids are present in one cells. So the amount of eGFP being synthesizes are high. But as cells divided, less copies are remained until it reach the point of no more plasmid present due to degradation and the only eGFP are the one from the integrarated in the genome. So the signal is much weaker than that of when the transfection first started.

Hello again,

just a clarification reading previous comments. You added that you have a single promoter for both eGFP and antibiotic resistance. I assume they are linked by an internal ribosome entry sequence (IRES) or self-cleaving peptides (T2A/P2A etc.).

In my experience this makes more difficult having resistant cells which are not eGFP+. Theoretically both proteins are translated from the same RNA so it's impossible to have one without the other. This plasmid topology should ensure that antibiotic resistant cells also express the eGFP. However you might have eGFP expression below the detection limit in most of the cells. This largely depends on the number of genome integrations per cells.

Generally speaking, due to their enzymatic nature in most cases, antibiotic resistant markers are effective in selecting stable integrants even when they are expressed at low levels. The same is not true for eGFP, for which you need a certain number of protein molecules for detecting a meaningful signal above the background.

This however doesn't change the downstream experimental approach. Regardless if the lack of eGFP is due to transgene silencing or low copy number, you'll have to keep antibiotic selective pressure and sort eGFP+ cells to establish pooled or (better) single cells clonal population.

Hope it helps

Francesco

One additional point concerning using the IRSE system. It is almost impossible to have a 1:1 ration of synthesis of both proteins. Usually one protein is expressed at high level while the second protein is expressed much less.

Dear Francesco Aulicino and Ngoc Toomey , apology that I understood wrongly.. After checking my plasmid structure again. Yes , my eGFP is dependent on CMV promoter for expression while my antibiotics resistance gene is dependent on another promoter to express. So what you guys have mentioned are correct.

The problem I have now is that the transfected cells survive in the selective media condition while lose the eGFP signalling over time. My lab doesn't have FACS facility.

Is there any other way to prevent the loss of the eGFP signal over time?

Many thanks.

Hello again,

If you don't have a FACS facility you can still isolate single cell clones by using end point dilution. There are two ways to do this:

1) make serial dilutions of your cell suspension after trypsinization until you reach ideally the concentration of 1 cell per 200 microlitres. At this point seed them in a multiwell 96, 200 ul per well. Let the cells untouched for 5-6 days without changing the media. At this point clones should be visible and can be inspected for GFP expression at the microscope. Always keep antibiotic selection and expand the eGFP+ clones. At each passage inspect eGFP expression and discard clones that become negative. Start at least with 2-3 mw96 plates. Many wells will be empty and some will contain mixed clones starting from more than 1 cell.

2) seed 100 cells in a 10 cm dishes. Maintain antibiotic pressure and let the cells grow without changing the media until clones are visible. Inspect the cells for eGFP expression at the microscope and pick eGFP+ clones using manual picking or cloning cylinders. Expand as in point 1.

Both approaches will work only if, despite silencing, a fraction of your antibiotic resistant cells is eGFP+. If by now your pool does not express eGFP at all you will have to start from scratch.

Hope it helps

Francesco

Dear Francesco Aulicino ,

Thanks for the suggestion on limiting dilution. We tried before and it seems like the single cell also loses the eGFP expression while it was still on the 96-wells plate.

The generation of the stable clones is really time-consuming and like you said, once it does not express eGFP, we need to start from the beginning (transfection) again..

If you attempted this previously and all of your clones lost eGFP than I would consider other integration methods. At best I would say that CMV is not ideal for your target cell line but cannot be sure of that. It is however known that CMV tends to be silenced over time.

Try to consider lentiviral vectors or changing the CMV promoter to CAG or hEF1a. However since both approaches requires additional cloning I would go for the lentiviral one. There is additional techniques which are quite good as well and do not require viral vectors suhc as PiggyBac transpositions or SleepingBeauty transposition. Maybe it's worth checking into these systems as well in your case.

Hi Francesco Aulicino ,

Thanks so much for all the suggestions and recommendations. Just to ask for lentiviral vectors, will it cause similar problem like what we are facing (the transduced cells lose the exogenous genome over time, in short time)?

We are also considering using transient transfection (oligonucleotide) for our downstream experiment to study some molecular pathway, do you think this will be an ideal approach as well?

I would recommend changing the promoter as well to EF1a just to clear CMV promoter silencing problems.

Lentiviral backbones are integrated completely so there will not be the risk of having the resistance integrated but not the gene. Most of the antibiotic resistant cells will be eGFP+.

All right. Thanks so much for the recommendation and suggestion! =)

I second Francesco suggestion of go the lentiviral way.

Suggestion: get a eGFP (HIV based) from SBI ( systembio.com ). Get pMD2.G and psPAX2 from Adgene for package to 293T cells.