Clonase reaction). I sequenced the plasmid and results from sequencing were positive for my gene. I went
Is the GFP in a bicistronic plasmid? If so IRES expression can be weaker that cap dependent expression. Is it possible that your GOI can have an inhibitory effect on GFP expression?
I guess you use higher cells, because you want to establish a stable cell line. You could isolate RNA from transfected cells, convert the mRNAs into cDNA and then use GFP specific primer to check for gene transcription.
But first I would check the reading frame as Thirupugal and Evelina suggested.
Can you explain how you are adding the GOI and GFP to your cells? Transient transfection of plasmids or a viral vector? Like Daniel Fullen said, if you are using an IRES-GFP cassette, the GFP expression can be quite weak especially if your cDNA is larger than 1kb. A better alternative is to use a T2A fused sequence of GFP and your GOI. If you are co-transfecting plasmids, and hoping for spontaneous integration, the efficiency of two integration events is low, and you'd need to use different antibiotic selection cassette on each plasmid (your GOI and your GFP expression cassette).
1. Did you confirm western blot analysis using antibodies against your POI as well confirmatory via anti-GFP?
2. Your GFP may be out of frame, however, if you saw protein expression in your Western's at the correct MW then it is most likely not out-of-frame and may be related to: conformation. Specifically, you GOI may undergo conformational folding that masks your GFP epitope during immunofluorescence staining but once linearized via SDS-PAGE becomes accessible to the primary antibody.
Thank you each and every one of you for answering.
@Arjan: I am positive my empty plasmid (without my GOI) has eGFP since whenever I do a transfection with the empty vector , I see strong GFP expression 48 hrs later.
@Evelina: Since I performed the transient transfection with a bunch of controls, (2 of these controls are the exact same plasmid with a diff GOI and they all are positive for GFP, so I believe the microscope settings are fine.
@Daniel Cohen:I received pre made PINDUCER 22 plasmid (with GFP ) from our collaborator and I did an LR reaction to shuttle my GOI (which was in a pDONR) into the Pinducer plasmid. It is an IRES GFP cassette and my GOI is 3.5KB
@Josh: I have not checked for GFP on my western yet. I am def. considering it as a last resort before I have to start from scratch again (and that would suck)
Ash- this is almost certainly a failure of the IRES-GFP cassette. It just isn't a good option for large cDNAs; I've seen this problem frequently in lentiviral cassettes that have the cDNA followed by IRES-EGFP. The best work-around solutions are to clone GFP in frame with your cDNA (you can use a T2A sequence if you don't want to have your protein GFP-tagged) or to add a second promoter between your GOI and upstream of GFP. I've had good success using the PGK promoter. Good luck!
Ash- I think there is some context-dependence as to how well the IRES sequence will work. But the problem is that I don't think the physical basis is known; it either works well empirically or not. I don't think there is anything wrong with your plasmid. If you are in doubt, try to sequence the IRES-GFP cassette portion to make sure it was unaffected by the recombination reaction.
I'll repeat some of what other people before me have said. Basically, is your GFP sequence in separate promoters than your gene of sequence or not? If not then it is possible it is cleaved during ER entry or exit. If yes some of the other suggestions might also check the problem. If different promoters, then it is possible for whatever reason, that your GFP is degraded or not expressed as strongly. It happens, I've seen GFP adenovirus having strong expression but GFP + gene of interest, the GFP expression to be far weaker. I can't say why, but in practice it happens. It doesn't mean your gene of interest is not expressed, it was always meant as an indication of expression. In any case, you should check your gene interest.
Hope that helps before I can help more with more information.
Ash- are you transfecting the pINDUCER plasmid or using lentivirus made from the this system? GFP will be much stronger in the context of virally transduced cells than transiently transfected cells using this plasmid. The likelihood of getting correct transcriptional initiation at the Ubc or Ef1a promoter in the context of the plasmid is low; mostly transcription will initiate from the strong 5' LTR promoter. Once the cassette integrates into the genome as a lentivirus, the second gene cassette can initiate gene transcription more independently. Hope this helps!
Common all too common a problem. Dan's responses are close to mine. I have all but given up on the IRES style reporter system. When it works, it is great, but even that can be a bit of black magic. You can try to sort for the GFP cells to get the strongest which has helped in the past. Otherwise, change vectors or co-transfect.
Have to agree to with Daniel, Thomas and David: Here is a 2012 paper outlining this and others with this problem !! http://www.ncbi.nlm.nih.gov/pubmed/23076521 In this case it actually hindered transcription of GOI. !!
If you have a strong promoter like CMV upstream of the IRES, one should expect you that production will be biased to the stronger promoter - more binding starts, faster consumption of precursors etc...
Is there any caveat if I want to use my lentiviral plasmid to overexpress the gene of interest without producing lentiviral particles?
Thanks
Susanna
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