why use beta galactocidase as a positive control in co-transfection - why not an antibiotic resistant plasmid (Dec/12/2008 )
Hi all,
Can anybody tell me why people still use beta galactocidase as a positive control when performing contransfection with another plasmid
The reason i ask is that we are trying to transfect a luciferase plasmid (with no selection marker) into our recently immortalised cells
As most people seemed to use beta gal we also tried this but when trying to work out if a cell has taken up both plasmids we first have to kill the cells and the results only show a percentage of how many cells have taken up the plasmids and we are still left with a mixed population of cells which requires a lot of subculturing etc to isolate specific cells with beta gal plasmid
Can anybody tell me why people dont use an antibiotic plasmid with no other insert (such as pBABE-puro www.addgene.org/1764) for selection instead?
Surely this is a more efficeint technique as non transformed cells will die on exposure to puromycin and removes subculturing etc?
Maybe i am wrong and this way is what most people are doing already but if it is i have not seen it mentioned in any recent papers
Is this beacause most plasmids would have selection markers already inserted and so positive controls are not needed?
Or is their some other reason such as beta gal being pretty kind to the cells in terms of differentaiation than an antibiotic resistant plasmid would be?
Any help/advice/suggestions would be great,
Cheers,
Cotchy
The B-gal assay show you what proportion of your cells have been transfected, you can assume that this proportion is the same as have been transfected with your other plasmid. From this you can deduce/extrapolate the levels of signal from a 100% transfected population, and use the proportions to normalise between experiments.
The vast majority of transfections performed are transient transfections. If you want to do a stable transfection then you use a plasmid with a selective marker (in this case you only need one plasmid) and select for the transfected cells. This process takes a fair while, anywhere between 3 weeks and several months to transfect, select and finally culture to a level where you have enough cells to work with, and then you have to characterise the cells to see that there haven't been any biochemical, morphological and growth rate changes between the selected and parent lines. A transient, non-selected transfection doesn't have these issues so it can be performed quickly and cheaply.
hi Bob 1 thanks for your reply.
I am pretty new to this whole area and do not fully understand everything you said in reply, if you dont mind could you explain some points for me a little further
Firstly what is a transient transfection?, i can figure out from the name what it should mean but i dont know how you can insert a plasmid into the genome which then removes itself later?
Also
You say to do a stable transfection i would need a selection marker and only one plasmid and this would take weeks to months to perform, how is this?
Firstly i have to use two plasmids as the plasmid i want to insert (a luciferase plasmid which has a promoter region specific for an oestrogen receptor binding site) has no selection marker therefore that is why i was asking if i could use an antibiotic marker plasmid instead of B gal as using this way i would be left with alot less starting cells/clones of cells which i could then trypsinise seperately into seperate flasks
If I pressume that at least one of the clones have taken up both plasmids, then after culturing each cloned colony seperately for a week or so to build up cells i could do some analysis to see if any clones have the luciferase and if so, then all the cells in this flask would probably have it, which i think would make for better results rather than the way you mention of working the resuslts on a percentage basis of cells that might have it
Is this what you mean when you say it could take weeks to months - i.e. culturing the cells to make up a large workable stock?
I have to do characterisation on the cells both before and after my transfection anyway so this does not add any more work to my project
Can you tell me what you think of using antibiotic plasmid apposed to B gal now?
When you transfect cells and analyze them in the few days later, you do transient transfection. the plasmid doesn't integrate the genome. when the cells divide, there is no guarantee that they will keep the plasmids, and after several divisions, the number of cells that still have a plasmid decay. If you want to perform long-term experiment with your transfected cells, you need to perform stable transfection. The difference is that there is a selection marker on the plasmid, so the cells that loose the plasmid will be killed by the selection reagent. Then all the cells that survive must have the plasmid and should express your protein of interest.
the beta-gal is only used to know how many cells you managed to transfect.
If you have 80 percent of cells that have incorporated the beta-gal you can assume that 80 percent of the cells have incorporated the plasmid of interest.
the beta-gal is only used to know how many cells you managed to transfect.
If you have 80 percent of cells that have incorporated the beta-gal you can assume that 80 percent of the cells have incorporated the plasmid of interest.
Ok Missele, thank you, i get it now! a transient transfection means the plasimd has inserted to the cell but not the genome and can be lost after a few dividsions, and does not change the characterisitcs of the genome/cell.
Justone other question on the original post, can using an antibiotic selection plamid also work?
i.e. Is it possible that B gal is also taken up into the genome or does it not have integration genes? (sorry as i am quite new to the area and dont know the terminology)
If it can also integrate into the genome then im sure it will change the characterisitcs in a similar way to the antibiotic plasmid integration and then using the antibiotic plasmid makes it easier to select?
Sorry if i asking questions you have already answered its just we would like to develop a cell line with 100 % of the cells having the luciferase and using B gal doesnt seem to allow this
Many thanks, Cotchy
in a stable transfection, the plasmid is not integrated in the genome (unless you use virus vectors), or am I wrong ?
you just force the cell to keep the plasmid by killing the one that don't keep it .
In your assay, I don't see how you will be able to keep your plasmid of interest. after a while you might loose the plasmid of interest even if 100 percent of the cells will still have the selection marker plasmid because you select these cells, and not the cells containing the plasmid of interest.
first of all, congratulations on your 1000st post Missele!
But I'm afraid I have to correct you on this matter. When you do a stable transfection, you also get incorporation of the plasmid in the genome of the cells. In some cases, people use autonomously replicating plasmids, but this is very rare, most of the time people prefer the stable integration in the host genome.
As for the luciferase setup, I would also combine it with a antibiotic resistance gene if you want to make a stable cell line. The b-gal cotransfection is just meant as a measure to estimate transfection efficiency in transient transfections, this is handy if you have lots and lots of different set-ups that do not require the production of a stable cell line.
good luck!
As i said i am pretty new to this and could be wrong as i assumed the plasmid did incorporate as:
Firstly we had to immortalise our (granulosa-ovarian cells) cells using another plasmid, psv3-neo which we did, this plasmid did have selective marker-neomycin which was used to select the cells, which resulted in a single clone only which took up the plasmid (only one small area from the whole dish had growth after treatment with neomycin) these cells have remained immortalsed for many many passages now which have all come from the one original cell
so on this info i pressume the plasmid must be inserted in the genome for it still to be expressed many passages later, (pSV3-neo works by knocking out tumour suppresoors genes such as but not limited to p53 which cause the cell to continuosly grow).
Secondly the next plasmid we need to insert is as mentioned, a luciferase gene which has a region specific for the oestrogen receptor,
We intend to use these cells for analysing compounds that can elicit the oestrogen receptor (ER). Basically if these chemicals or Endocrine disrutping chemicals (EDCs) as they are also known can excite the ER they will then turn on the luciferase gene as it has inserted directly down stream in the DNA which is regulated by the ER so both will be turned on simultaneously and thus light is given off if a chemcal can interact with the ER
Sorry for the long explanation but going on the techniques/procedures above am i right to pressume that a plasmids DNA can integrate with the host DNA? otherwise our luciferase would be of no use as it would not integrate in the region we want it to?
To dpo . thanks. just one more question. there is no particular sites on a plasmid for the integration of the plasmid in the genome, am I right ? so any plasmid could finally be integrated anywhere into the genome ?
thanks both of youfor your help, but as we did not develop this luciferase plasmid ourselves i do not know much about it and cannot alter it to contain an antibiotic resistance as we do not have ownership or the capabilities
but seemingly it has a strecth of DNA that is complimentray to a fragment of DNA located on the genome downstream of the DNA for ER so i think it will incorporate here, do you think this is possible and is correct dpo?
my main source of knowledge on this topic, my supervisor, is away until the new year and i cannot ask her but i am sure she knows exactly what happens to the plasmid
is this techniques also used in knockout gene ananlysis incorporation at an exact point?
on the other hand however for our immortalisation plasmid we have no idea where this had inserted and i do not think we can tell either.