1. Can anyone explain to me what to expect when comparing the overexpression of the gene and subsequent protein from lentiviral plasmid during transient transfection and transduction. Are the levels of overexpression dependent only on copy number of plasmids/inserts or there are other important factors involved?
What orders of overexpression are typical for transduction?
In my experiment transient transfection gives about x1000 overexpression after 24hrs and transduction leads to 10-40 overexpression after 5 days. This is not only the effect of the differences in the efficiency.
2. Could anyone tell me when the gene and protein overexpression reaches its plateau after transduction? In my experiment it seem to be 4-5 days post transduction. Is this typical? The protein has no degradation sequence tag and the expression of mRNA is driven by PGK or UBC promoter.
3. Does anyone have the experience with PGK driven expression in lung cancer cells? It seems to be weak- much much weaker than from UBC promoter.
1) Transfection allows lot more plasmids to enter a cell, while transduction typically allows one or two copies of the proviral DNA to enter a cell. Therefore, unless you infect with extremely large quantities of virus, overexpression by transient transfection is always more efficient than transduction. However, I have not quantified how much of overexpression occurs. It will also be dependent on cell type.
2) For 293T cells, reporter gene expression driven by a CMV immediate early promoter plateaus at around 20 hours post transduction. I do not know what cell lines you work with and the plateau could be different with different cells and different promoters and different genes and also upon codon optimization of the respective gene.
3) I have not worked with those cells and hence, do not know the answer to that.
The answers given above assume that you are using an easy-to-transfect cell line. With cells that are difficult to transfect, you will barely get any plasmid entry and sufficient levels of over-expressed protein. Therefore, you will invariably have to use lentiviral transduction (and antibiotic selection). Also you may have to clone the cells and screen for levels of your protein of interest.
First thing first.... Viral transduction using lentivirus vector is far more efficient and successful way of expressing a gene of interest than simple lentivirus plasmid transfection (as they are mostly large in the range of 10kb in size, excluding your gene of interest... thus very hard to transfect) on any given day for any cell line including stem cells.... if transfections were so great, then why the heck we even bother to look for potentially kiss of death gene delivery system of lentivirus vector...!!!???? like a lot of precaution of handling those bugs and executing the transduction in lentiviral dedicated room with BSL2 protection etc.... The biggest plus for lentivirus transduction is that you can CRANK UP the concentration of virus per unit volume and increase the MOI from 1 to 100 particles per cell, so the probability of integration of gene under CMV-promoter per cells can sky rocket and you get the stable high expression in almost 100% of cells, whereas transfection can anywhere between less than 1% (in hard nuts...) to 70% (in easy guys like 293T...) with the best combination of cells and reagents used...... So lentivirus transductions are the best way to go. The efficiency of generating a stable cell lines with transduction is far efficient than the one you can get with transfections, so unless their is some inevitable reasons to do transfection over transduction, if anyone doing simple transfection to express any gene when they can DO the lentivirus prep and transductions... then they need to go back to basics.... Thinking you can increase the plasmid load to each cells more than a virus particle is ABSOLUTELY WRONG......!!!! rather it can be opposite as you can do multiple infections or high concentration virus particles....
The only plus with transfection over transduction comes from the future use of stable cell lines generated from these two methods.... the viral transduced lines are usually no, no to be taken to the FACS analysis (in some places...) or injected in to MICE for sure.... as it has a probability and can generate active virus particles in the mouse (far fetched but possible...) whereas cell lines from transfecyion do not have the helper plasmids exposure thus have a very low probability of producing any virus particle.....!!!!! In the end if these are not the limitations then the lentivirus transduction is the way to go......
Dr. Rajabi,
I would like to respectfully tell you that you are digressing from the question. I believe the question is about protein expression and not about stable cell creation.
Yes, lentiviral transduction is efficient in "creating" stable cell lines (as you rightly pointed out) depending on the cell line. But for protein over expression, transfection helps quite a bit, in easily transfectable cell lines. If you can electroporate your cells, and depending on the future use of the cells, transfection will definitely give you better plasmid intake, and in most cases, higher protein expression.
You may not be able to electroporate stem cells , nor could you possibly transfect them. In those cases, lentiviral transduction is the only way to go, ( as you aptly mentioned).
There is a plateau to plasmid uptake by transfection and there is also a plateau or limit to virus uptake by the cells during transduction. Whether plasmids can be overloaded into cells or whether lentiviruses can be overloaded into cells is something I do not know of, and I am sure there are no absolute comparisons for the same that have been published so far. Hence, calling one superior over the other for overloading cells, is in my opinion, a wrong comparison. Furthermore, beyond a point, regardless of how much you overload the cells with whichever method, you will not be able to boost protein expression or any other function in the cell, as the translation machinery, once maxed out, possibly shuts down leading to cell death.
As for FACS analysis, you may fix the cells and run them on flow cytometers and there are multiple papers published by using flow cytometers to measure infectivity, where the lentiviral genome encodes a fluorescent promoter to report a successful infection.
I am certainly willing to go back to basics to check if I am right or wrong, but it seems to me that hard words and comments have to be backed up with solid experience or proof.
In response to you, since your comment talking to me (whereas I did not do that.... when I responded earlier and that is not the purpose of this Q & A is) only thing I want to tell that I missed it in the first reply was..... Lentivirus transduction distant to go to nucleus immediately and get integrated to the genome of all kind of cells and can generate mRNA and Protein far efficiently and forever (if stably selected to make cell line...) whereas plasmids have much lower efficiency of doing that.
As for as your argument of "you have not seen any such publication comparing these two techniques" so let me tell you, if someone trying to publish a comparison of transfection and lentivirus transduction will indicate that the person has plenty of free time to waste and wants to do something... irrespective of significance of doing that....!!! And finally no more arguments... you believe that you are right then be like that, I am not forcing you to agree with me... neither I am forcing Tomasz to agree with me... I just gave my opinion based on my own knowledge and use of techniques..... I may tell you that; I do all my experiment while standing on my head instead of feet..... it doesn't mean you have to do the same and agree with me.... it is just a place of discussion and not to prove who is right or wrong.......... I may be totally wrong, so what... it works for me.....!!!!
Tomasz,
I think that your reason for expressing or over-expressing a particular gene (or genes) in a cell is an important consideration for the answer to your question. Transient over-expression is useful if you want to maximize gene product in a cell to test its effect - you can opt for high copy number and high production. If you want to produce the protein permanently, then it is much better to create a stable transfection in your cells to create a cell line that will grow and continually produce protein for study in that cell or for collection in cell lysates. If you are seeking stable insertion, copy number is somewhat limited since it depends on the number of insertion sites in that cell type for your particular insertion sequence. Once you have defined the problem, you will have your answer as to which is preferable. Then it is only a matter of technique to optimize for your cells and what makes for good insertion sequences.
Thank you all for the comments specially to Sanath who tried to answer my specific question. Hasan Rajabi - of course there is a specific reason why I am using lentiviral transduction - I need stable cell lines with different levels of overexpression because it seems my protein "shows its effect" after a few cell divisions and yes I do have fluorescent marker to easily monitor the level of overexpression. As I am new to this method and quite used to simple transfection I was surprised by the fact that there is so big difference in the levels of overexpression and for me it is reasonable that this difference is not only dependent on the differences in copy number - or only on that? Thanks to Christofer Duntsth I am now sure that the levels that I have obtained are quite typical. The other thing that surprised me is the fact that my overexpression after transduction reaches its plateau in 4-5 days - but this probably because of the slow accumulation of fluorescent marker in mitochondria and/or its degradation during mitochondria quality control (it is mitochondria targeted DsRed2).
Can you please explain to me the problem with FACS sorting of lentiviral transduced cell cultures. Of course I use BSL2 conditions to transduce and maintain the transduced cell cultures. It is known that FACS can produce a lot of aerosol that can contain possibly oncogenic virus - I believe this aerosol is the concern?. But the half-life of this virus is very short - what are the safety reasons for abandoning sorting - lets say after two weeks of culturing and few passages? I have not read about it in two biosafety handling informations and paper that I obtained. Am I not aware of a possible danger?- this is important - please explain.
Hi Tomasz,
There are some reports that the so called "multiplication incompetent virus particles" when transduced to certain cell lines can regain the power of generating virus particles and this probability goes much higher if you injected the cells to a mouse who is carrying the latent pMLV gene. This was first reported to happen with cells transduced with Retrovirus vectors but later found to be true for lentivirus vectors too.
I will strongly recommend you to test the supernatant of the cells which are transduced and selected with drugs, (so that can be called as free of all "active virus particles") for the pVSV gene. If you have GFP in your virus particle then I will suggest you to add that culture media to 293T cells and wait for 48 hours or so before selecting with the same drug you have in your virus particles and if you have any visible number of cell left over and they show GFP positive under fluorescent microscope then you know you have the problem otherwise not. On the other hand if your FACS machine is sitting inside "laminar flow hood" (as here in DFCI we have one machine dedicated to sort transduced cells) then you don't have to worry about the whole process that much. Here is a reference to explain what I am talking about
Articles
Laboratory Safety for Oncogene-Containing Retroviral Vectors
John T. Gray*
St. Jude Children’s Research Hospital, Memphis, Tennessee,
Applied Biosafety, vol. 16, No. 4, 2011 (absa.org)
Hope these information helps in your research.
bets of luck.
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