stable transfection - (May/09/2008 )
Hi all,
Please can anyone help:
I need to know whether you need to keep the selection antibiotic in the media once you have a stable (from a single clone) cell line growing? I'm unsure, as some poeple are saying there's no need and others are saying yes to be safe?
You have to select cells for antibiotic resistance continuously.
Please can anyone help:
I need to know whether you need to keep the selection antibiotic in the media once you have a stable (from a single clone) cell line growing? I'm unsure, as some poeple are saying there's no need and others are saying yes to be safe?
there is no need if you have selected a stable clone or mix of clones;
if a continuous application of selecting antibiotics is recommended: Is there a higher risk to lose the stably transfected gene than any other parental gene?
The way how I was taught is that you keep the selecting antibiotic but in a lower concentration.
You do need to select continuously but at a lower antibiotic concentration. There are several reasons:
1. The integrated transgene has more chances of being silenced (methylation etc.); the cells in which it is silenced will be removed by the continued antibiotic.
2. If some cells loose the expression of transgene, and they have a growth advantage as compared to the cells with transgene expression, you will loose all stable cells in a few cycling times if antibiotic is not added.
3. If any wt cells contaminate your stable line (at the time of picking clones or later), they will be killed.
4. If you had not selected a single clone, but are working with a selected pool, then there are chances that some wt cells may have barely survived, and they may outgrow the stable cells later.
--
On the other hand, if you don't worry about wt contamination, have selected a clean clone, and passed through several cell splittings, you can check out an aliquot over a few passage to see if transgene expression does not go down (always freeze down several vials of original before doing this), if so, you can use stable cells without selection.
HTH
1. The integrated transgene has more chances of being silenced (methylation etc.); the cells in which it is silenced will be removed by the continued antibiotic.
2. If some cells loose the expression of transgene, and they have a growth advantage as compared to the cells with transgene expression, you will loose all stable cells in a few cycling times if antibiotic is not added.
3. If any wt cells contaminate your stable line (at the time of picking clones or later), they will be killed.
4. If you had not selected a single clone, but are working with a selected pool, then there are chances that some wt cells may have barely survived, and they may outgrow the stable cells later.
--
On the other hand, if you don't worry about wt contamination, have selected a clean clone, and passed through several cell splittings, you can check out an aliquot over a few passage to see if transgene expression does not go down (always freeze down several vials of original before doing this), if so, you can use stable cells without selection.
HTH
answers 2.-4. are well described but is it true that a genome-integrated, f.i. by recombination, foreign gene has a higher chance of methylation and deletion? Does anyone know a paper which analyzed this?
in any case, one has to take a lower passage number if higher passage numbers lose their quality
in any case, one has to take a lower passage number if higher passage numbers lose their quality
it is true. Cells have natural mechanisms to silence foreign genes (particularly viral, transposons, prokaryotic etc, which we use as tools) for their own sake. But...this information i have picked up through informal channels and don't know of any particular paper; i suspect there must be a classic paper followed by some more studies. I wish somebody can provide such reference..