Hi all,
We have a lentiviral vector with a gene of interest that we would like to express. There is no requirement for stable cell line construction, so we will only be transfecting transiently. However, our most proliferative cell line is not HEK, and therefore I'm curious for transient purposes, if the lentivector could be expressed in other cell lines, e.g. CHO?
I see some other posts on RG, e.g. https://www.researchgate.net/post/Why_are_293_HEK_cell_used_as_viral_vector_production_cell_over_other_cell_lines_What_advantage_does_it_have. But again to our interest, we're not producing the viral particles.
Our viral vector consists of an LTR followed by a CMV promoter before our gene of interest. Would this suffice for transient transfection? Or would binding to LTR limit CMV binding in other cell lines? Thanks for any advice.
Hi Leran,
Transient transfection will "work" for any cell line but the efficiency will be very low to the point where no positive cells will be observed/sorted.
I've tested several transfection reagents on different cell lines and only HEKs yielded a sufficient amount of positive cells. Any other cell line would have a 1-2% transfection efficiency and the few positive cells would end up being overgrown by non-transfected cells.
Usually, higher amounts of transfection reagents will increase the efficiency but not by much.
You could also test different transfection reagents. Although most work on the same principle with lipo-particles, I've seen different outcomes even when using different generations of the same product ie. lipofectamine 3000 vs 2000.
Kajs Hadžić
Thanks for the reply Kajs!
Interesting observation, may I ask if you are using a lentiviral or a regular mammalian plasmid? I do not experiment heavily in transient transfections, but I used mammalian (CMV) plasmids in HEK and CHO cells and they both yield decent transfection efficiencies. HEK for sure had a higher efficiency. I haven't used lentiviral vectors for this purpose, hence the question.
This lenti we're using is a commercial vector and in the instructions the company claimed transint transfection would work well in HEK. But I'm not sure about other cell lines. And CHO cells are not really used for anything related to lentiviral production / transfection.
I'm wondering if you have any insights on why there is a wide difference among cell lines?
For lentivirals, do you know if transcription factors bind to the CMV? How might LTRs interfere?
I have used lentiviral vectors for transient transfection in many human cell lines (HEK293, A549, HeLa, etc) and in all cases I saw a high expression of my gene of interest. As long as your gene is expressed from a promoter (like CMV) and not expressed via the LTR, then you should get good expression. I typically have used Lipofectamine 3000 for these experiments, but I see similar results with other transfection reagents.
I have not used CHO cells so I can't comment on whether you might see different results in those cells.
Hi Leran Mao
I mainly worked with the PWPI mammalian plasmid from Adgene https://www.addgene.org/12254/ a high copy plasmid with an Ef1a promoter and usually we inserted the genes over the PmeI site, upstream of IRES.
It was the protocol that was somewhat established in the lab when I took over. If you're interested contact me via email and I'll gladly send you the entire thing as well as a troubleshooting guide.
Generally, we used HEK-293FT for viral particle/titer production since lentiviral infection is more efficient than transient transfection and for our purposes, we usually didn't care much about the integration of the inserted plasmid into the DNA of the cell line.
I couldn't specifically tell you why there are differences between cell lines but I remember reading at some point that it has to do with the permeability of the cell membrane. Some cell lines have "denser" cell walls and are therefore less prone to larger particle infections. HEKs, especially the FT variant are used for titer production because of the SV40 large T antigen and their general protein production efficiency.
In addition to infection vehicle size, one thing that will determine efficiency is the charge of the particles/DNA. That's why polybrene is used to neutralize the charge on the DNA fragments and allow for higher infection efficiency.
Again, this is not directly related to transient transfection as most of my actual lentiviral work consisted of lentiviral particle infections.
Hope this helps somewhat.
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