Needs of advice for Stable Cell Line Preparation

Frank O. Fackelmayer Frank at Fackelmayer.de
Thu Aug 29 05:26:29 EST 2002


Dear Khoa,
here are some comments to your questions

>        My intention of the work is  to get a stable cell line from a
> single clone containing the desired DNA sequence. With the attempts to
> achieve such a stable cell line, i initially prepared a construct with 2
> inframed - Myc - tags at the C-terminus of the DNA sequence. This
> construct is maintained in pcDNA3 plasmid. Then, after confirming for
> the activity and protein expression of the newly made construct. I used
> lipofectamine to transfect the construct into raw 264.7 cells (mouse
> macrophages). This cell line is one of its kind for transfection !!! The
> transfection efficiency that i had was only about 1 %. (Electroporation
> is recommended from the literature, however, the electric shock is
> highly toxic to cells and it might disrupt the DNA of the construct,
> thus affecting the activity of the encoded protein).


Cell lines differ very much in their transfectibility. Often, suspension
cells are much more difficult than adherent cells. I strongly recommend
to optimize the electroporation. Its outcome also depends on the cell
line, but it is not too difficult to optimize the electric parameters; a
crude determination is possible with some 5 different settings. Use
empty EGFP vectors for these experiments, and you can easily see the
proportion of transfected cells. Usually, there is a voltage where the
cells look perfect but are not being transfected, and at a higher
voltage they are all dying. It is quite common that good transfection
efficiency is seen in a narrow range just before cell death is too
pronounced (e.g. 50V lower). 
Also, try to vary the amount of DNA in your transfection. Too much will
also kill the cells. For us, 10ug of DNA per 100mm plate of cells is ok
(transfected in 800ul of complete medium).
The DNA will NOT be disrupted by electroporation!


> 
>    The ratio of DNA to lipofectamine is 1ug : 4ul, 2 ug :4ul, 4ug :16
> ml, 5 ug : 16 ml , 6 ug :16 :ml , 7 ug : 16 : ml and 8 ug:16ml.
> Protamine sulsulfate was also added to enhance transfection efficiency.
> 
> 
>     Two days after transfection, the cells were diluted and selected
> with 418 (400ug/ml) for transfected cells. A lot of cells were killed
> and the remaining cells were then collected and maintained. However,
> Western Blotting results  show a very low level of protein expression in
> these Raw cells in comparison to a positive control of DNA/Myc
> transfected CHOP cells (relatively significantly high transfection
> efficiency).

it is common that the expression in stable cell lines is way lower than
with the same construct in transient assays. A low but detectable amount
is NOT a negative result.

> 
>      Due to the surviving ability with G418 exposure, the cells were
> still kept and further diluted to screen for positive sclones from the
> single colonies. Here immunofluorescent miscroscopy was used after
> staining the cells to determine the positive clones ( half of the cells
> from each clone were taken for immunofluorescent analysis and the other
> half were maintained). This work has been carried out for quite a while
> with repeated dilution and colony collecting, but until now a stable
> clone is yet to be available ...

the low amount of protein may not be detectable in immunofluorescence.

> 
>     My questions are:
>    - Why are the nontransfected cells resistant to G418 at such a fatal
> dosage ?

several facts have to be considered:
* integration only occurs with linear DNA. When you use supercoiled
plasmid for transfection, some plasmids will be linearized by cellular
enzymes, and will be integrated. However, opening of the plasmid can
occur anywhere in the plasmid, and can easily disrupt your gene but not
the resistance gene. This risk increases with the length of your insert.
For a 3kb fragment in a 6kb construct, the chance to ruin your gene is 50%.
* antibiotic resitance is conferred by an enzyme. Even low amounts are
suffient to allow a survival of the cells. The same amount of protein
without enzymatic activity may not be detectable, eg. by western blotting.
* your construct (but not the resistance gene) is inactivated because
the cell "feels" that your protein is somehow not good. Only cells with
downregulated expression will propagate and form colonies. Inactivation
can occur by different mechanisms. e.g. deletion or methylation of the
promoter. In such cases, an inducible system should be used.


>    - Would there be an alternative (s) to single out the positive
> clones?

Cell sorting into multiwell plates is a good method when you have access
to such a machine. A prerequisite is, however, that there is something
that you can detect in the sorter without killing the cells.
Fluorescence, e.g. for GFP fusion proteins, is great.

>    - Overall, are there some potential problems in my preparation for
> the stable cell line?

the number of positive clones you can expect is directly dependent on
the transfection efficieny. With 1% as you said, there is very little
chance that you will get well expressing clones unless you have a really
large number of plates to start with. So, optimization of your
transfection is the primary goal first.



Hope this helps
Best wishes for your experiments,

Frank



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