Protein expression help needed - Mammalian cells (Dec/03/2007 )
Dear all,
I have tried to transfect my plasmid DNA (pcDNA4/HisMax) containing viral glycoprotein gene into Vero cells for the past few months. However, my attempts always ended with failure of expression. I could not see any significant difference on the protein profile on gel and I could not detect the recombinant protein on Western blot.
For my transfection, I am using Lipofectamine 2000 and I modified the protocol by referring to several past comments on this forum. However, nothing seems to work for me.
Can someone please share with me your experiences in expressing protein in mammalian cells? Theoretically, it sounds very straightforward and easy but practically it is very tough.
Many thanks in advance.
Cheers.
Have you tested with an EGFP vector to see what % cells are transfected? I would encourage you to try 293 or its derivatives, such as 293T or 293FT, cos-1 or cos-7 cell lines. these are more transfectable.
or tell us what have you done step by step see how much we can do about it.
I agree with genehunter about using 293 cells or their derivatives for protein expression. They are easy to transfect also.
Also what promoter is driving the expression of the transgene in your plasmid?
Many thanks for your replies.
The pcDNA4/HisMax expression vector is a overexpression vector that is driven by CMV promoter.
For my transfection, the following is a brief summary of my procedure.
1. A day prior to transfection, 1x106 cells/mL of cells were seeded in a 6-well plate.
2. 10uL of lipofectamine was added to OptiMEM (to 100uL).
3. 4ug of DNA was diluted in OptiMEM (to 100uL).
4. Both components were incubated for 10 min at room T.
5. Both were mixed and incubated further for 30 min at room T and topped up to 1mL with OptiMEM.
6. 500uL of the lipid-DNA complexes were added to cells. Alternatively, I aspirated the media prior to the addition of 1mL of the complexes and 1mL of OptiMEM was added to the cells 6 hours post-transfection to prevent the media dry out from the plates.
7. Cells were harvested 24 and 48 hours post-transfection by scraping off the cells in PBS.
That is how I did my transfection. Please do comment if I have done it correctly. Thank you very much.
Cheers.
You have done mostly correct.
Would 10^6 cells/well too much? what is the % of confluency, like 70-90% full?
lipofactamine 2000 may be a better choice, although I am not 100% sure for this cell.
I like the alternative way in #6 than the 500 ul protocol, coz, you only added 1/2 of the amount.
Again, have you checked your transfection rate with a reporter gene that can be either examined under fluorescence microscope (EGFP), or stained (such as beta-Gal)?
Is there a particular reason for you to stick with vero cells?
Would 10^6 cells/well too much? what is the % of confluency, like 70-90% full?
lipofactamine 2000 may be a better choice, although I am not 100% sure for this cell.
I like the alternative way in #6 than the 500 ul protocol, coz, you only added 1/2 of the amount.
Again, have you checked your transfection rate with a reporter gene that can be either examined under fluorescence microscope (EGFP), or stained (such as beta-Gal)?
Is there a particular reason for you to stick with vero cells?
Again, thank you for your reply, genehunter.
I have determined the number of cells to be seeded a day prior to transfection. With that number of cells, the confluency will be about 80% full the next day. Further, most of the time the cells are not distributed evenly onto the surface.
No, I have not really transfected a reporter gene into Vero cells. I tried it once with pEGFP-N2 plasmid and performed a Western blot by detecting the GFP protein with anti-GFP monoclonal antibody. However, I failed to detect the protein. I do wish I have a fluorescence microscope in my lab, unfortunately not even one is available.
After that, for every of my transfection, I did include a positive control from my expression vector, which has a LacZ gene. Again, I blotted the protein onto NC membrane and detected with anti-His antibody. However, no product was detected. Only when I left the blot in the substrate for nearly 30 minutes, I would see an extremely faint band of about the LacZ product size (very faint til my senior questioned would that be my illusion). Since I have to leave the blot for nearly 30 minutes, I hardly convinced myself that was the product as I also got all the unspecific bands on the blot by then. Meanwhile, I also found that after the transfer, there were still lots of high molecular weight proteins remained on the gel when I stained it with Coomassie blue.
So, I drew a conclusion that probably my protein was expressed at very low level and due to the low transfer efficiency, I couldn't detect my protein on my blot.
The reason I expressed my proteins in Vero cells was that so far most of the literature expressed the proteins in Vero. In addition, Vero cell is widely used to isolate the virus I am working on and some study showed that some cell-lines can not be used to isolate the virus and also to express the viral proteins. Therefore, for the safe side, I just started with Vero and ended with lots of problems.
Try to use lacZ gene and 0.1% gultaldehyde fixation, X-Gal staining 4-24 hrs at RT to see how many cells turn blue. This will give you some idea about what % transfection rate you have. It seems to me your transfection efficiency is very low. You may want to use either lacZ or luciferase reporter plasmid to do a series of optimization experiments using a fixed amount of DNA and varied amount of transfection reagent. Both reporters are very good for quantitaive purpose. LacZ is less sensitive than luciferase, but is cheaper to run.
Can you also test if Cos cells serve for viral production of the virus that you are studying. You can infect these cells with wild type virus and test for the titer. Cos cells are much easier to transfect. You should have no problem to get >50% transfected with lipofactamine 2000.
After adding the complex, you kept the cells in OPTIMEM till the harvesting?
Sure that your Vero cell is good? Not too old? Did any other in the lab also do transfection into this cell line? What are their results (if have)? What about other labs? Ask for some good Vero cells and try again? Also, performed parallel transfection into 293 cells (GFP is good) and see if it is due to your transfection reagents' problem.
Sure that your Vero cell is good? Not too old? Did any other in the lab also do transfection into this cell line? What are their results (if have)? What about other labs? Ask for some good Vero cells and try again? Also, performed parallel transfection into 293 cells (GFP is good) and see if it is due to your transfection reagents' problem.
Yes, I left the cells in OPTIMEM with the complex til harvest.
I totally not sure how good is my Vero cells but I obtained it from a research institute, where the cell has been used for propagating viruses.
Another labmate of mine has tried to express my protein in the cell, and he failed too. However, he was able to express his own protein of interest in the cell, and he succeeded though the expression level was very low. So, I can't complain much about my cell and my PI will never accept this type of reason.
Sure that your Vero cell is good? Not too old? Did any other in the lab also do transfection into this cell line? What are their results (if have)? What about other labs? Ask for some good Vero cells and try again? Also, performed parallel transfection into 293 cells (GFP is good) and see if it is due to your transfection reagents' problem.
Yes, I left the cells in OPTIMEM with the complex til harvest.
This may not be very good for the cells. Is this the standard protocol???? I think that it is not so in the protocol for Lipofectamine transfection. After incubation in OPTIMEM for about 4h, you can either change or add complete medium (DMEM, is it, for Vero?) with FBS in for incubation till harvest, usually about 16-24h later.
You and your labmate used GFP as control and didn't see good transfection? In 293?