Scientific Report HPRN-CT-2000-00089
Titration of DNA and transfection reagents using several transfection systems in
different cell lines:
For titration fo DNA and transfection reagents and evaluation of other conditions for
transfections (media, buffer…) two cell lines were chosen that have very different
characteristics. B16F10, which is an adherent murine melanoma cell line and
K562, which is a human leukemia suspension cell line. K562 cells can be
stimulated to express human transferrin receptor prior to transfection. These cells
therefore can be transfected in a receptor mediated attempt using human
transferrin (Tf) coupled transfection complexes (Tf-PEI25, Kircheis et al., 2001).
Since production of BAC DNA is expensive and time consuming due to the low
amount of only one single copy of DNA per bacteria, in a first aproach cells were
transfected with a luciferase expressing reporter plasmid (Planck et al., 1992) or a
GFP (green fluorescent protein) encoding plasmid (pGFP-N1, Clontech) to
evaluate whether cells were highly transfectable with one or the other transfection
system (data not shown). Promising systems were then adopted to use of BAC
DNA.
The BAC used in these studies (EBV BAC, Baker et al., 1997, see Fig. 1) encodes
eGFP as marker gene and to ensure episomal replication and nuclear retention in
human cell lines additionally the viral elements ori-P and EBNA-1. The size of the
vector has been increased to 150 kb by the insertion of a 130 kb human DNA
fragment (position indicated by arrow head).
Fig 1. Bac used in these studies
In Fig. 2 a summary of transfections carried out in B16F10 cells is shown.
Compared to the preliminary transfections with plasmids (data not shown) one can
say, that all transfection reagents that successfully transfected plasmid were also
suitable to transfect cells with BACs and additionally, that the most efficient
transfection systems using plasmid or BAC were the same.
B16F10 transfected with PEI22 (N/P 7,5), precomplexation with PEI2 (N/P 2,8)
10,00
8,00
8,00
% transfected cells
% transfected cells
B16F10 transfected with PEI22 (N/P 5), pre-complexed with PEI2 (N/P 2,8)
10,00
6,00
4,00
2,00
6,00
4,00
2,00
0,00
0,00
0,5 µg DNA
1 µg DNA
2 µg DNA
3 µg DNA
4 µg DNA
5 µg DNA
4 µg DNA
5 µg DNA n. d.
0,5 µg DNA
1 µg DNA
2 µg DNA
3 µg DNA
4 µg DNA
5 µg DNA
4 µg DNA
5 µg DNA
4 µg DNA
5 µg DNA n. d.
B16F10 transfected with PEI22 (N/P 7,5)
10,00
% transfected cells
8,00
6,00
4,00
2,00
0,00
0,5 µg DNA
1 µg DNA
2 µg DNA
3 µg DNA
B16F10 transfected with PEI25 (N/P 7,5)
10,00
10,00
8,00
8,00
% transfected cells
% transfected cells
B16F10 transfected with PEI25 (N/P 5), pre-complexed with PEI2 (N/P 2,8)
6,00
4,00
2,00
6,00
4,00
2,00
0,00
0,00
0,5 µg DNA n. d.
1 µg DNA
2 µg DNA
3 µg DNA
4 µg DNA
5 µg DNA
0,5 µg DNA n. d.
2 µg DNA
3 µg DNA
B16F10 transfected with Lipofectamine Plus
10,00
10,00
8,00
8,00
% transfected cells
% transfected cells
B16F10 transected with Lipofectamine 2000
1 µg DNA n. d.
6,00
4,00
2,00
6,00
4,00
2,00
0,00
0,00
0,5 µg DNA
1 µg DNA
2 µg DNA
3 µg DNA
4 µg DNA
5 µg DNA n. d.
0,5 µg DNA n. d.
1 µg DNA
Fig. 2. BAC transfections in B16F10 cells (n.d.: not done).
2 µg DNA
3 µg DNA
In Fig. 3 the results obtained with K562 cells are summarized. Again transfections
with plasmid allowed to determine the most efficient transfection systems, which
could be confirmed in transfections with BACs. In this case, receptor mediated
transfection systems were more effective than all other transfection reagents.
K562 transfected with PEI22/TfPEI25 (N/P 7,5; 4+1)
5,00
4,00
4,00
% transfected cells
% transfected cells
K562 transfected with PEI22/TfPEI25 (N/P 5; 4+1), pre-complexed with PEI2 (N/P 2,8)
5,00
3,00
2,00
1,00
3,00
2,00
1,00
0,00
0,00
0,5 µg DNA
1 µg DNA
2 µg DNA
3 µg DNA
4 µg DNA
5 µg DNA
0,5 µg DNA n. d.
5,00
5,00
4,00
4,00
% transfected cells
% transfected cells
2 µg DNA
3 µg DNA
4 µg DNA
5 µg DNA
4 µg DNA
5 µg DNA
4 µg DNA
5 µg DNA n. d.
K562 transfected with PEI25/TfPEI25 (N/P 7,5; 4+1)
K562 transfected with PEI25/TfPEI25 (N/P 5; 4+1), pre-complexed with PEI2 (N/P 2,8)
3,00
2,00
3,00
2,00
1,00
1,00
0,00
0,00
0,5 µg DNA n. d.
1 µg DNA
2 µg DNA
3 µg DNA
4 µg DNA
0,5 µg DNA n. d.
5 µg DNA
K562 transfected with Lipofectamine 2000
1 µg DNA
2 µg DNA
3 µg DNA
K562 transfected with mit Lipofectamine Plus
5,00
5,00
4,00
4,00
% transfected cells
% transfected cells
1 µg DNA
3,00
2,00
1,00
3,00
2,00
1,00
0,00
0,00
0,5 µg DNA n. d.
1 µg DNA
2 µg DNA
3 µg DNA
4 µg DNA
5 µg DNA
0,5 µg DNA
1 µg DNA
2 µg DNA
3 µg DNA
Fig. 3. BAC transfections in K562 cells.
All transfections were carried out using 5 x 105 cells per well with the indicated
amounts of BAC DNA and were repeated several times (in Fig. 2 and 3 means of
all
transfections
are
dedicated).
Transfection
systems
using
PEI22
(Polyethylenimine, 22kD, MBI Fermentas, Germany) turned out to transfect more
cells than any other transfection system. In B16F10 cells more than 9 % of the
cells expressed the respective marker gene using PEI22, in K562 cells over 4,5 %
of the cells expressed GFP when PEI 22 is combined with TfPEI25 in a ratio of
4+1. It is important to mention, that the amount of DNA which is necessary for
optimal transfection efficiency is at least 3 to 4 times higher using BAC DNA
compared to plasmid but is finally limited by toxic effects of DNA itself or
transfection reagents which have to be scaled up according to the higher amount
of DNA used for transfecions. For this reason not all amounts of DNA were
examined with every transfection system, but only the amounts of DNA were
analysed that were supposed to be effective after having done the transfections
with plasmid. Additionally, it could be seen that transfections with BAC DNA lead to
two (B16F10 cells) to ten (K562 cells) times lower numbers of transfected cells
even under optimized conditions compared to transfections with plasmid.
Another problem that had to be solved was that the high amount of BAC DNA
which is necessary for best transfection efficiency may cause problems during the
preparation of transfection complexes like formation of visible precipitations in the
transfection mixture e. g. by the use of 6 µg BAC DNA and PEI25. These
precipitations can be prevented by pre-complexing BAC DNA with PEI2 prior to
adding PEI 25 or PEI 22 without reduction of transfection efficiency and in the case
of K562 cells pre-complexing Bac DNA enhanced transfecion efficiency with both
transfection systems (PEI22/TfPEI25 and PEI25/TfPEI25). These facts are of
special interest especially for BACs which are even larger than the one used in
these studies.
In summary one can say, that the milestone of delivery of BAC DNA into cell lines
using a variety of different transfection systems which was set for the first 12 month
has successfully been reached.