Download Transfection of Eukaryotic Cells Using the DOTAP Method – The

F E A T U R E S
Transfection of Eukaryotic Cells
Using the DOTAP Method –
The Rapid Screening Route to
the Heart of Your Cells
K I R S T E N I . Z E N Z , R . L A U S , V. E C K S T E I N , A N D W. M U L L E R - R U C H H O LT Z
I n s t i t u t e o f I m m u n o l o g y, C l i n i c o f t h e C h r i s t i a n - A l b re c h t s U n i v e r s i t y, K i e l ,
Michaelisstrasse 5, D-24105 Kiel, Germany
Introduction
During a study on the mapping of cell surface molecules, we looked for a procedure by
which large numbers of transfected cells could be evaluated on a routine basis. Such a
method had to be practicable in terms of the time and materials required. The target cells
were to be transfected with fusion constructs containing the transmembrane regions of different proteins and a soluble extracellular portion of a marker protein. The soluble extracellular portion can be detected on the cell surface since it is inserted and held at the cell
membrane by the transmembrane region. The level of expression of the transfected
proteins was determined by fluorescence labeling with antibodies against the marker protein. Cells were initially transfected by electroporation, and the analysis carried out on a
flow cytometer. For comparison, parallel transfections using DOTAP were performed and
analyzed by fluorescence microscopy.
Materials and Methods
COS-7 cells (SV40-transformed kidney fibroblasts from the African green monkey) were
transfected with fusion constructs containing randomly chosen parts of a human cDNA
library from T-lymphocytes and a sequence coding for the extracellular portion of CD 31 (1).
Transfection was first carried out by conventional electroporation: 1 x 107 cells were
electroporated with 10 µg DNA, incubated in 10 ml culture medium (IMDM with 25 mM
Hepes and 10% FCS) for 60–72 h, and subjected to flow cytometry. A conventional fluorescence histogram printout served as documentation.
For comparison, 6 x 104 cells were transfected with 0.2 µg DNA using DOTAP
Transfection Reagent in the method described by Boehringer Mannheim. The cells were
incubated with the DOTAP/DNA mixture for a period of 6–16 h in IMDM containing no
serum. The culture medium was replaced by fresh IMDM (with 25 mM Hepes and 10%
FCS); 24 h later (i.e., 30–40 h post-transfection), the marker protein on transfected cells
was labeled with a fluorescent antibody. These transfections were evaluated by microscopy;
nonetheless, a semi-quantitative analysis regarding the frequency of positive cells and the
intensity of fluorescence was performed.
In an initial study, 21 fusion constructs were electroporated into the COS-7 cells,
followed by flow cytometric analysis. In subsequent series of analyses, 40 clones were
transfected in parallel by subjecting them to either electroporation or the DOTAP method.
Subsequently, 261 fusion constructs were screened using the DOTAP method. Forty-nine
of these (i.e., those molecules showing a high degree of expression), were subjected to
analysis with the FACScan.
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NO.2 [1995]
F E A T U R E S
Results
Molecules exhibiting a high degree of
expression could be reliably detected using
either fluorescence microscopy or flow
cytometry. Analogous results were obtained
using both methods where the same fusion
constructs were used in parallel. However,
there are apparent differences between the
methods: the time and materials required,
and the ease with which results can be
interpreted.
With electroporation and flow cytometry, quantitative results concerning the
frequency of transformed cells and the
fluorescence intensity of individual cells
within a population of transfectants
(Figures 1 and 2) can be obtained. However, the following points suggest that this
particular method is not suitable for
routine screening of large numbers of
transfected cells:
(i) Electroporation consumes large quantities of materials per experiment:
10 µg DNA and 1 x 107 cells (equivalent to the yield of one 10 cm culture
dish) are necessary in order to obtain
reasonable results.
(ii) Because of the time required for performing electroporation of COS-7 cells
with subsequent flow cytometry, a
maximum of 16–20 transformations
can be performed in parallel.
Figure 2 Transfection by electroporation. Flow cytometry; 14% positive cells;
High fluorescence yield.
the intensity of fluorescence in individual
cells. In addition, the method allowed the
definite localization of the fusion protein
(outer cell membrane, membrane system
of the cell compartments, or nucleus).
Figure 3, a fluorescence micrograph of an
entire cell, shows that the fusion protein is
expressed in the entire cell, including the
cell membrane and processes. Figure 4 is a
fluorescence micrograph of a transfectant
containing a protein expressed strongly
within the area of the nuclear membrane
but not in the cell membrane. In addition,
the secretion of certain molecules can be
clearly determined, as can be seen in
Figure 5.
Figure 4 Fluorescence micrograph
of a COS-7 cell transfected using
DOTAP. The fusion molecule is expressed in
the area of the nuclear envelope but not in the
cell membrane.
ments can be performed with one plateful
of cells) and 0.2 µg DNA (1/50th of the
material required for one electroporation).
The only disadvantage of the fluorescence microscopy method is that it is
somewhat time consuming to determine
the expression rate. However, with practice, this also can be quickly estimated to
within ±10%.
Figure 5 Fluorescence micrograph
of a COS-7 cell transfected using
DOTAP. Expression takes place in the entire
cell, with secretion into the extra-cellular space.
Conclusion
Figure 1 Transfection by electroporation. Flow cytometry; 24% positive cells; Low
fluorescence yield.
Figure 3 Fluorescence micrograph
of a COS-7 cell transfected using
DOTAP. The fusion molecule is expressed
throughout the entire cell, including the cell
membrane and processes.
The DOTAP/fluorescence microscopy
method has a number of advantages over
electroporation and subsequent flow
cytometry. Evaluation by fluorescence
microscopy enabled classification according to the frequency of positive cells in the
entire cell population and also according to
Another advantage of the DOTAP/fluorescence microscopy method over electroporation/flow cytometry is that the former
requires smaller quantities of cells and
DNA. With DOTAP, between 64-80 parallel transformations can be performed, each
requiring only 6 x 104 cells (160 experi-
BIOCHEMICA
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NO.2 [1995]
Overall, the transfection of eukaryotic
cells using DOTAP and subsequent analysis by fluorescence microscopy is a valuable method when large numbers of transfected cells have to be analyzed. Further,
the time and material requirements are
minimal. In addition, valuable information
can be obtained as to the distribution of
the molecules within the cell. ■
Product
Transfection Reagent
(DOTAP)
Cat. No.
1202 375
Size
5 x 400 µl
See page 17 for local pricing.
References
1. Stockinger, H. et al. (1990) J. Immunol. 145:3889.
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