Please help with stable transfection
dk at no.email.thankstospam.net
Wed Oct 2 21:40:21 EST 2002
Khoa.Ngo at baker.edu.au ("Khoa Ngo") wrote:
> I am in the middle of the preparation of a stable transfectant clone
>with RAW264.7 murine macrophage cell line. For which, a source of about
>50% positive stable transfectant cells was used for a limiting dilution
>with a ratio of 1/3 cell per well in a 96 well plate. Non-transfectant
>cells, used as supporter cells, were added together with the
>transfectant cells to the wells in medium without G418. After a few
>days, G418 was added to kill the supporter cells and in about 10 days,
>colonies were clearly seen under microscope for about 30 wells. From
>these, only 13 wells had colonies visuable to the naked human eyes, thus
>cells from each of the 13 wells were screened for positive clones.
>However, fluorescent microscopic observation (after immunofluorescence)
>shows some head-aching results for me. It is supposed to show either non
>or all cells are positive, but non of the clones could demonstrate a
>complete batch of positive cells, most of the clones have a few positive
>cells, while some show approx. 30 - 40 % positive cells, which is less
>than the original source (a sample of the original source was screened
>to confirm this lesser effect).
> Here, i am wondering if someone can tell me what could have
>happened in order to lead to such results? and what would be the posible
>options available to get the expected clones?
IMHO, most likely nothing has happened and everything is normal.
Pretty heterogenous (over)expression of exogenous genes is almost
a norm in most stable clones. You can get one of this positive clones,
subclone and observe the same clonal variations in second generation.
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