A549 cells (adenocarcinoma lung cells) were transferred with organelle marker plasmid to achieve expression of different organelles within the cells. In total I had 4 different stably transfected cell line. Transfection was carried out by first optimizing the concentration of G418 (selection antibiotic), then growing cells with selected concentration over 3 weeks before picking colones.
Stably transfeccted clones were confirmed through confocal microscopy because plasmid DNA is GFP tagged. Transfection was done last year August, result suggested Transfection worked.
At the moment, stable cell line seems to have lost the expression of GFP, I'm so worried to what would have gone wrong and how it's possible. Cells are still been mainted in G418 at a lower concentration.
Any idea idea on what to do because when viewed with confocal, you can see very few cells still expressing GFP and tried using cell sorting FACSaria to collect GFP cells, this wasn't succesful.
Thank you in advance for your replies.
Hi Abiola we have A549 cells but I haven't tried generating stables from them.
However the short answer is yes - over time all cell lines will begin to lose GFP expression if cultivated long enough due to selective pressure in culture (cells expressing large amounts of GFP are at a growth disadvantage compared to cells expressing low or no GFP). This is one of the reasons that, even from cells grown from a single clone, GFP levels will be variable in the larger, expanded, cell population. Maintaining the cells under selection helps stop this but even then non-GFP cells will appear - either caused by the cells deleting the GFP protein (but leaving the ab resistance gene) or silencing the GFP reporter (see: "Effects of Epigenetic Modulation on Reporter Gene Expression: Implications for Stem Cell Imaging", Krishnan et al 2006). One way to stop the cells deleting the GFP is to use a vector where the GFP and the antibiotic resistant gene are driven from the same promoter (such as an IRES vector).
Generally the loss is quite small (occurring over many passages) but dependent on cell type it can actually be quite rapid (as we saw when we ourselves were looking at CMV driven GFP/RFP in the same H9C2 cells used in the paper link above, whereas a line using HEK293 cells, generated at the same time, had stable fluorescence over > 5 passages).
How rapid was the fluorescent loss? After generating a stable transfectant I would always bank some cells as soon as possible (as a master stock) - but I guess you have not done this?
Regardless of transfection any cell line should never be continuously passaged in culture as phenotypic drift will occur (always generate a Master and Working banks, perform experiments then get new cells from the working bank for more experiments).
Depending upon the % of GFP cells remaining you might try a limiting dilution approach (but if the % was too low for cell sorting it will probably be too low for this to work).
Sorry I can't be more help,
Gary
You should always try to have as many aliquots as possible from the original line. More passages leads to decreased expression and this is true of many cell lines. I remember a previous laboratory I was part of having a lot of difficulty with A549 transfections.
Dear Abiola, to obtain stable transfected cell lines, two steps are critical. First step is to use retroviral vector for transduction (such as MSCV or lentiviral vector). Second step is recloning of transfected cells several times. By this way we obtained mouse cell lines with stable expression of GFP. We observed the loss of GFP expression only after long propagation of tumor cells in vivo under pressure of immune system in H-2d mice.
Please, take into consideration that a DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. In the following review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. We exemplify a number of studies, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. Finally, we conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes.
Please see the following manuscript for details:
Stepanenko AA, Heng HH. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat Res. 2017 Jul;773:91-103. doi: 10.1016/j.mrrev.2017.05.002.
Attila Lehotzky you say if driven by a CMV you may silence the GFP - do you have any details as to why this is?
Not have real details, it is said that CMV promoter easily silenced in cells. I think there are papers about it.
Best matches for cmv promoter silencing by methylation:
Promoter methylation and histone modifications affect the expression of the exogenous DsRed gene in transgenic goats.Nuo MT et al. Genet Mol Res. (2016)
Silencing of fat-1 transgene expression in sheep may result from hypermethylation of its driven cytomegalovirus (CMV) promoter.Duan B et al. Theriogenology. (2012)
Targeted methylation of CMV and E1A viral promoters.Hsu CC et al. Biochem Biophys Res Commun. (2010)
I do not really interest it as it is a reversible change in cells addign some drugs... see the last paper.
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