Is your gene normally expressed? Have you also checked if you have only single integration by southern blot?

I suggest you perform RT-PCR on the whole construct and sequence it to make sure everything is OK. You should also consider the possibility that some proteins do not tolerate any of the normally available tags at their C-terminus because they induce their mis-conformation. I guess you did have that information before proceeding to insert the tag in you GOI.

Hi Domina,

I forgot to mention that you should calculate the success rate of your integration. Normally the percentage is low. Because you don’t have a selection marker, I guess you are performing PCR on a mixed cell population which has both positive and negative integration. In this case, you should perform PCR with oligos encompassing a region outside the marker. You should obtain two products whose quantity may give you an idea about the percentage of cells bearing your construct.

In this case, I suggest you screen your cell under an fluorescence microscope and if you have some positive cells you should perform cell sorting with FACS.

fastest is to disolve you cells in RT-PCR buffer and do a crude PCR with primer specific for your transgene (alternatively there are also specialized medium for that from Ambion for instance

http://products.invitrogen.com/ivgn/product/4402953.

stratagene side-step (which I have tested and it works)

or from quiagen:

http://www.qiagen.com/Products/Catalog/Assay-Technologies/Real-Time-PCR-and-RT-PCR-Reagents/FastLane-Cell-cDNA-Kit#orderinginformation

these are for real-time PCR

Cells do not always express the complete transgene, so fluorescence might not be there or expression is too low.

In addition you should probably verify you construct using transient transfection to check that it is fluorescent (it happens that during the cloning procedure mutations occur )

Hi,

an alternative would be imaging based methods. most commonly used is mRNA FISH. Might help do detect transcripts if they are not very abundant.

Labeled probes are now commercially available:

https://www.biosearchtech.com/store/product.aspx?catid=224%2c318%2c323

http://www.ncbi.nlm.nih.gov/pubmed/18806792

I would say that RT-PCR or in situ hybridization would work to check the mRNA. Also the tag you are using might cause some issues with visualization. I have had problems with m-cherry (although it worked very well for others) when inserting it at the C-term vs. N-term. If the tag is toxic to your cells you should see fluorescently labeled cells floating in your cultures unless you are changing the medium before imaging. Also if it is a common tag you could check expression using DAB immunocytochemistry to your tag to make sure that your not just seeing very low expression in any given cell making it more difficult to visualize via fluorescent microscopy.

Thanks for all the responses - very helpful.

Mina

In situ hybridization could be useful to see the mRNA within the cells. However, RT PCR is the fastest way to detect the expression of the transgene. Chimeric construct of the inserted gene that contains a fluorescent tag could make your life much easier.

YOu should try checking the fluorophore by western blotting

If you are not routinely doing RT-PCR technically they would be challenging since you have to take care of RNA degradation while prepping and DNA contamination while PCRing. If you are a first timer to RNA, I would recommend northern since you will actually see the size of your RNA and will be able to see also how much it is expressed wrt WT construct. You will have to prepare RNA for northern too, but here you can check on a urea gel before proceeding for western

I am sorry , I mean t before proceeding for northern transfer

I'd also suggest RT-PCR (that's reverse transcriptase, not real-time). You can use Trizol to isolate mRNA (manufacturer's instructions are fine), and then use ImpromII or another reverse transcriptase to make cDNA. That cDNA is then used for PCR as you would use genomic DNA, and you'll be able to see if your mRNA is what you expect. Is there an insert between your GOI and the fluorescent protein? Make sure there's no splice site or stop codon between them. These things have a way of sneaking up on you! Also, I'd suggest expressing the plain fluorophore (without your gene fused to it) in your cells as a control for both the microscope and the fluorophore. Good luck!

Hello!

I agree with most things said above (not all, though).

I think you need to stimulate your cells for the expression of your GOI and then do fluorescence acetivated cell sorting to eliminate the non-transgenic cells. You just need literally a few cells to start off a new cell population from there. Prefer puritiy (high GFP expression) over quantitiy.

Anyway, you need to check your insert for correct integration (reading frame; stop codon). I recommend to PCR-amplify (with Pfu DNA polymerase, not Taq) and sequencing.

One naive question: are you sure, your GOI+GFP is expressed under your current cell culture conditions? If not, then there cannot be GFP expression either. This can be checked via RT-PCR, as recommended above. Personally, I wouldn't go for Northern.

If you have a positive RT-PCR signal but no GFP expression, then you most likely have a frame shift or a mutation in your GFP sequence.

Good luck!

Alexander, the GOI is definitely expressed in this cell line as our lab routinely blots for this protein. Also, its an essential protein that is expressed in all cells.

I'm going to purify the amplicons from the PCR validation and have them sequenced (don't know why I didn't think of that!)

The consensus appears to be that RT-PCR is the easiest and fastest way to determine if the mRNA is being expressed so I'm going to try that.

Thanks to everyone for all the helpful suggestions - much appreciated!

Mina