Low transfection efficiency/protein expression - I've tried everything! (Nov/17/2006 )
Thanks in advance for any help and advice and for reading this long question!
The plasmid encodes a secreted protein and should be detected in both cell lysate and media. The protein is FLAG tagged and the vector is pCMVtag. A previous postdoc got very high expression of the protein in both cell lysate and media as expected, however I cannot achieve this result using the exact same protocol.
I get very low expression in cell lysate and very low or nothing in media.
However the strange thing is using another plasmid (also FLAG tagged, in pcDNA3 vector) enocding a different secreted protein I can get high expression and see the protein in both cell lysate and media using all different transfection conditions.
I cloned the insert from the plasmid with low expression into the pcDNA3 plasmid and it did not improve the results.
I want to use 293T cells, and I also tried 293. I used low passage number cells and I tried different stocks of frozen cells.
I tried both Fugene 6 and Fugene HD. For fugene 6 I tried a 1:6 or 1:3 DNA:Fugene ratio. For Fugene HD I tried 2:3 and 1:8. I used a ug amount of DNA that was in the suggested range (for Fugene 6 it was 0.4 ug in a 24 well plate). I also tried using 1 ug of DNA.
I followed the Fugene protocols exactly.
I used various confluencys as suggested by the transfection reagent (~50%, 70%, 80%, and 90%). I also counted cells and used the amount suggested (I tried between 50,000- 200,000 for both transfection reagents).
I did not get a lot of cell death and the cells are negative for mycoplasma.
I cultured the cells between 2-5 days after transfection (changed to serum free media 1 day after transfection). The previous postdoc got high levels of protein expression on day 2.
I tried using 1% serum in OptiMEM instead of serum free for the 2-5 day culture. Otherwise the media is always DMEM/F12 (10% FBS) for the transfection and I tried with or without antibiotics (but always without antibiotics and serum for making the fugene/DNA complexes)
For the plasmid with high expression and the one with low expression I purified them the same way using Qiagen midi prep kit. I also tried using stocks of the plasmid purifed by someone else with no difference in results. A260/280 ratio was 1.8. I ran the DNA on a gel and it was not degraded. The DNA is in TE at a concentration of 0.5 ug/ul. Phenol/Chl extraction and EtOH precipitation after the midi prep did not affect the results for either plasmid.
Sorry this is REALLY long! I greatly appreciate any help and advice, I am out of ideas!!
Are you 100% sure about the sequence of your plasmid?
Seems that your transfection efficiency should be OK, as the other protein is in the media and lysate. Most likely, that DNA was extracted in the same way as the DNA you're struggling to get the protein from?
Is your protein for some reason extra sensitive to proteases (shouldn't be, because the other person got it, but who knows).
How did you measure expression in media and lysate? Blotting?
Thanks for your help, Varius.
I haven't sequenced the plasmid but I think I will check the insert sequence as you suggest, since the promoter is ruled out because it didn't work in the other vector.
I don't think the protein is sensitive to proteases, but I actually tried adding a protease inhibitor to the media (one that is okay for cell culture) and it didn't affect the expression levels. That is also why I tried using 1% serum for culture because I read that serum contains protease inhibitors.
The DNA was extracted in the same way and I am only using my preparation of the good expressing plasmid (I obtained a small amount from another lab and amplified it myself) while I have tried both my preparation and the stocks of two other postdocs for the low expressing plasmid. That is why I was wondering in my other question if some plasmids need very special preparation treatments to work for transfection.
I'm using western blotting with anti-FLAG antibody for detection and I detect both the high expression and low expressing protein on the same blot.
Thanks again for your help
I have two comments for you: 1. try to work with a construct carrying a reporter gene first, EGFP for example, for the optimization step (fixed amount of DNA with varied amounts of fugene); 2. be sure that your DNA concentration is right.
Thanks for your help, Genehunter-1.
I checked my DNA concentration by spec and also by running on the gel and compared it to the standard and both my plasmids looked the same.
I have an EGFP expression plasmid, but my question is if I test that won't it just show the transfection efficiency/expression level of that particular plasmid?
low transfection efficiency may happen for several reasons; as you did good optimization, it may not a problem by the protocol; when we do transfections with GFP, after 12h cells begin to express, so I would look earlier than after two days; may be the peak of expression has already turned
but expression is not only a matter of protocol; some proteins are "disliked" the cell as they block viability or physiology, and will not be expressed to a higher extent;
as genehunter-1 says, optimize with a GFP f.i. classical pEGFP, and follow expression after 8h by fluorescence, and than all 4-6h if it will be possible to show a time-dependent profile of expression
Thanks for your help, kosmodrom. I'm going to start a transfection today and I will try checking expression levels after shorter culture times.
I had a problem with transfection efficiency because my DNA was contaminated with tRNA that wasn't fully chewed up by the RNase. You could run your DNA on a gel again, but only when the marker gets halfway down the gel otherwise you won't be able to see the tRNA cause it's pretty small.
Have you tried the post-doc's stock of the same plasmid??
I would also try doing a single digest. One of my preps was resistant to restriction digest, and even though the DNA looked clean and I had the correct amount of DNA, I didn't get it to express in cells.
Hope this helps.