Hi all. I have one reporter plasmid and need to create a stable cell line. The plasmid lacks mammalian selection and I was planning to build one in. A colleague however suggested that co-tranfection with a second plasmid carrying resistance should do it as long as both are linearized. Is this correct?
Luca, I agree with some of the comments saying it is possible to do cotransfection. I have done this myself many times in the past, and it is far easier than constructing expression vectors that have the reporter and selectable marker directly linked. The critical factors to consider are:
(1) Both plasmids must be linearized at a single site that doesn't interfere with either's expression.
(2) The reporter/selection ratio should be 10:1.
(3) Cotransfection works best using calcium coprecipitation, but I've had success using electroporation and lipisomal methods.
(4) The drug concentration used to select clones should be titrated on untransfected cells beforehand, such that the concentration is 2-3x the amount that kills 100% of the cells. Using less will give you false positives; using more will kill even transfected cells. Serum source and concentration should not be varied until stable clones are generated.
(5) Once clones are selected, they should be screened for expression. Discard negative clones. However initially keep representative clones of all levels of expression, not just the highest ones. Transfectants should be cultured for a few passages and then re-analyzed. You may find some of the highest expressors have become negative because they took up too much DNA and were unstable. Depending on your experiment, you may want to save clones that express different levels to make quantitative functional comparisons.
(6) For long term culture, always use media containing the selectable drug at 1x the amount used above. At this point it is wasteful to use more, and these drugs tend to be quite expensive.
Much of this advice is also applicable when transfecting with single plasmids containing the selectable marker and reporter gene. Good luck.
Yes, it is entirely possible. The plasmids do not even have to be linearized, although linearization does increase the chance of a productive integration as the breakpoint will not be random. However, a significant proportion of your stable clones will not express your gene of interest (GOI), so you will have to screen your stable clones for expression of your GOI.
The problem is that the reporter will likely be expressed only in 1% of all cells .It carries a chromatin regions with an enhancer driving GFP, but GFP is expected only to occur in a small subpopulation of cells. I was under the impression than the vast majority of cells integrating one will integrate the second, since plasmids uptakes occurs in concameters. How easy is to clone a resistance cassette in?
Yes you can although we have empirically found more variability in expression of the transgene of interest, so you might have to screen more clones.
It is absolutely correct that transfection with 2 separate plasmids will yield some stable clones of the GOI. Those experiments were done 20 to 30 years ago. While it is true that some of the clones selected for antibiotic resistance will not include your GOI, most will for the reasons Luca has mentioned above. And yes you can subsequently screen for your reporter gene .But this is really the case for stable clones anyway. There will be varying levels of expression of your protein among these clones due to sites of integration. Using the 2 plasmid approachdoesn't change the steps that needs to be done in any stable transfection: first transfect and select for whatever the selectable marker is and then second screen these clones for expression level of your protein. There are always varying levels. Choose several clones to grow up and then freeze back as level of expression can change over time.
Add your plasmid in a 10:1 ratio with a second plasmid containing a resistance marker and co-transfect. This certainly works if you choose Ca phosphate co-precipitation as your method of transfection because both plasmids form a mutlimer conglomerate (called a pekeliosome, I think) so your resistance marker is physically linked to your plasmid of interest. This ratio should also work for electroporation (10 parts your plasmid, one part resistance plasmid).
I've created stable cell lines using co-transfection of a GFP-reporter plasmid and a linearized hygromycin marker (Clontech catalog #631625). Each step should be optimized, from determining the ratio of marker to plasmid DNA (see Clontech's literature) to choosing the level of hygromycin selection best for your cell type. The selection of GFP-positive monoclonal progeny populations can be cumbersome. Make sure you are able to demonstrate expression of GFP in the expanded monoclonal cultures by another method other than fluorescence microscopy, so you don't get tricked by autofluorescence. I used immunoblots against GFP. This also helped me find the strongest expressors.
This selection process will take more time than you think (and requires diligently attending to your selected clonal lines). Given that, you still may want to consider building the resistance marker into the same construct, in which case, I would suggest a GFP expression cassette derived from Clontech's bicistronic vectors like pIRES-hyg3 (catalog #631620).
It is not a good idea. Most of stable cell you get will be reporter+resistant gene or resistant gene only. I would do a one more construction work and insert resistant gene into first plasme. Linearized plasmid before transfection, because I don't want random linearized by in cell enzyme.
Hi Luca,
I may have misunderstood your question so let me know if im completely off track.
I would personally clone a puromycin selection marker in for fast selection. You could clone a 2A FMDV sequence downstream your GFP followed by PURO.This way you ensure same levels of expression of both cDNAs. I have a PURO-2A-GFP which works well. It can take a couple of weeksto make but once you have the cassete made is nice to have because yuo can replace PURO or GFP for your gene of interest depending on what you want to do.
A high ratio of selection to reporter is the standard approach. My approach is to go through the selection process (weeks to months depending on the cell line's characteristics), use cloning rings to select individual colonies, then characterize a representative number of clones (several to a dozen). I also learned to culture the cells without selection media during assays. Although the reporter could be lost from a stable line over time it would take several passages for revertants to dominate the culture. The absence of the selection media tends to improve the performance of the cell line and thus the quality of the data.
Luca, I agree with some of the comments saying it is possible to do cotransfection. I have done this myself many times in the past, and it is far easier than constructing expression vectors that have the reporter and selectable marker directly linked. The critical factors to consider are:
(1) Both plasmids must be linearized at a single site that doesn't interfere with either's expression.
(2) The reporter/selection ratio should be 10:1.
(3) Cotransfection works best using calcium coprecipitation, but I've had success using electroporation and lipisomal methods.
(4) The drug concentration used to select clones should be titrated on untransfected cells beforehand, such that the concentration is 2-3x the amount that kills 100% of the cells. Using less will give you false positives; using more will kill even transfected cells. Serum source and concentration should not be varied until stable clones are generated.
(5) Once clones are selected, they should be screened for expression. Discard negative clones. However initially keep representative clones of all levels of expression, not just the highest ones. Transfectants should be cultured for a few passages and then re-analyzed. You may find some of the highest expressors have become negative because they took up too much DNA and were unstable. Depending on your experiment, you may want to save clones that express different levels to make quantitative functional comparisons.
(6) For long term culture, always use media containing the selectable drug at 1x the amount used above. At this point it is wasteful to use more, and these drugs tend to be quite expensive.
Much of this advice is also applicable when transfecting with single plasmids containing the selectable marker and reporter gene. Good luck.
If you want to construct a new plasmid with both resistant gene and the reporter, you can link these two genes with the P2A sequence ( 69 bp)
Thank you all for the helpful comments. Just few details. My reporter sequence will drive GFP expression in only 1% of cells even when integrated in 100% of population. The reporter guide the expression of a gene epigenetically controlled and expressed only in a small subpopulation. The PURO marker will drive Cherry. My hope now is to select for Cherry and then screen genetically for GFP in several clones. Insertion of a cassette in my reporter gene seemed like a good idea, but the problem is finding plasmid donors that have an easyly tranferrable PURO or HYGRO cassette (including promoter and polyA for the cassette). Ideally any vector that has the entire cassette between StuI, PsII NotI or HindIII should make my life easier!
In My opinion it should work fine enough following Gerald's suggestions. My guess is that if it is a reporter vector the fact that you might have one not carrying the reporter every million that does, will not affect the results of your experiments. However, it is also true that if you plan to do this more often, it might be a good investment to try to obtain a PURO resistance backbone, in which to clone the reporters. It will speed up your work and reassure your results.
Go ahead with it. Linearized both, take 1:5-10 target:resistance ration, do not care about the method of transfection (phosphate, liposomal, electroporation) and everything will be fine in 8-9 resistant clones out of 10. Good luck!
Please read from the beginning iA la. All is explained. Antibiotic resistance gene is in second plasmid and both are transfected into cell
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