hi,
Iam facing a weird problem after transfection. I am using C3H10 meschencymal cell line which is a stable clone expressing a protein which is GFP tagged. Iam using this cell line to transfect another protein having a red tag. both the protein has G418 resistance. After a month, I have few colonies. But when I see it under the fluorescent microscope the resistant cells neither have GFP or red tag.
1 . How is this possible, when I have used a stable clone with a GFP protein ( I did check the cell for fluorescence before the experiment.) for transfection.
2. Do the cells loose the protein for any reason.
Suggestions are welcome
it is also possible that expression of GFP is silenced. This phenomenon depends on the type of promoter in the plasmid that was used to transduce the cell line. CMV is a commonly used promoter that is prone to silencing.
it is also possible that expression of GFP is silenced. This phenomenon depends on the type of promoter in the plasmid that was used to transduce the cell line. CMV is a commonly used promoter that is prone to silencing.
Recombination events between the DNAs derived from the two plasmids is possible too, since they are both G418 resistant and have common sequences. Thsi could result in a non-sense recombinant mRNA. Perhaps use plasmids existing different antibiotic resistance genes (hydgromycin, for example).
The promoter might be methylated. It is a common mechanism to shut down the ectopic expression of a protein/miR that the cells does not like.
Thanks Rongqing. Is there a way out for this or can I prevent the methylation.
(1) Use FACS to sort out the GFP high cells for further transfection.
(2) When you get your "RED" cells. Freeze down a batch of them (low passage). You might grow your RED cells for 1/2 month to see if the RED signal would fade away.
(3) If so, you could use your low passage cells or use FACS to sort out GFP high and RED high cells for your experiments.
Good luck!!
Dear Jaya,
You SHOULD LOOSE your protein in at least 95 colonies from 100 after 3-6 weeks of cultivation in case you use plasmid vector with CMV or similar promoter. CMV plasmids transfection provide 95-99 percent of so called transient expression which is not genomic one, so it cannot be stable. To obtain stable transfected cell lines it is better to use lentiviral vectors. However they have their drawbacks in selection process and are not very presentative because of target protein relatively low expression.
One thing like vector construction is also important. Fusion of fluorescent protein and your target one under the same promoter normally supress the target one production significantly compared to FP you see under microscope which is cloned first in vector.
Thank you for the suggestion. The stable clone was made before I came to this lab. I will see the vector map for further details.
I will also check to make sure that what is being called stable is really stable, which means the vector is incorporated into the genome.
This is due to genomic instability of stable cell line.Which I feel a great disadvantage. Some times stable cells will lose the gene while they are dividing so in these cases you cant see the gene of interest which you cloned. To avoid that you freeze the stable cells at the beginnig and store it in liq nitrogen.you revive one vial at a time.
all the best
If this happened you can only transfect/infect cells newly, make colonies selection and produce new stably transfected line. Double-transfectants are even less stable. After several years of exercising with different vectors and attempts to obtain stable transfectants (not unsuccessful but not efficient enough for in vivo works) I started to make results in vitro within 3 weeks of first experession when it is combined transient-genomic and high enough. Mr. Reddy's advise to freeze stable transfectants before expression is lost is very useful.
Greetings
hi. May be your clones were not stable. You can try Limiting dilution a week after transfection. May be you need to optimize transfection protocol. You can also use MAR sequence in you vector, which helps in stability of clone.
Best of luck
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