Journal of Experimental Botany, Vol. 57, No. 12, pp. 2985–2992, 2006
doi:10.1093/jxb/erl065 Advance Access publication 26 July, 2006
RESEARCH PAPER
HaloTagTM: a new versatile reporter gene system in
plant cells
Christina Lang, Jutta Schulze, Ralf-R. Mendel and Robert Hänsch*
Institut für Pflanzenbiologie, Technische Universität Braunschweig, Humboldtstraße 1, D-38106 Braunschweig,
Germany
Received 20 March 2006; Accepted 23 May 2006
Abstract
HaloTagTM Interchangeable Labeling Technology
(HaloTag) was originally developed for mammalian cell
analysis. In this report, the use of HaloTag is demonstrated in plant cells for the first time. This system
allows different fluorescent colours to be used to
visualize the localization of the non-fluorescent HaloTag
protein within living cells. A vector was constructed
which expresses the HaloTag protein under the control
of the 35S promoter of cauliflower mosaic virus. The
functionality of the HaloTag construct was tested in
transient assays by (i) transforming tobacco protoplasts and (ii) using biolistic transformation of intact
leaf cells of tobacco and poplar plants. Two to fourteen
days after transformation, the plant material was incubated with ligands specific for labelling the HaloTag
protein, and fluorescence was visualized by confocal
laser scanning microscopy. The results demonstrate
that HaloTag technology is a flexible system which
generates efficient fluorescence in different types of
plant cells. The ligand-specific labelling of HaloTag
protein was not hampered by the plant cell wall.
Key words: cLSM, HaloTagTM, particle bombardment, protoplasts, reporter gene expression.
Introduction
Labelling of proteins is an important tool in the study of
their functions and dynamics in living cells (Giuliano and
Taylor, 1998). The introduction of the fluorescent protein
GFP (green fluorescent protein) and its derivatives has been
a great breakthrough in cell biology. GFP is widely used as
an extremely powerful vital marker in a large number of
organisms (Chalfie et al., 1994; Sheen et al., 1995;
Zimmer, 2002), and labelling of proteins by genetic fusion
has extended our understanding of protein function in the
last decade. Fluorescent proteins as reporter genes have the
primary advantage that their in vivo assay requires neither
long sample preparation nor the uptake of exogenous
substrates, as compared with alternatives such as luciferase
or b-glucuronidase (Haseloff and Amos, 1995; for a review
see Hanson and Köhler, 2001). Moreover, these proteins
can be expressed and monitored within intact tissues, cells,
or cell organelles without any destruction of the material.
To this end, more than 40 variants of the original jellyfish
GFP have been constructed which fall into seven main
classes of excitation and emission spectra (Palm and
Wlodower, 1999). These different colours can be used to
study protein localization and interaction especially in colocalization experiments with double or multi-labelling of
cells. Here, the different GFP colours have to be fused to
the proteins of interest prior to the transformation experiment. Yet, experience shows that some of the proteins have
to be labelled with more than one of the GFP variants to
give a clear and distinguishable image under the confocal
laser scanning microscope (cLSM). The new HaloTagTM
Interchangeable Labeling Technology, developed for mammalian cells (Promega, Mannheim, Germany), introduces
a new flexibility to fluorescence microscopy. Instead of
a gene for a fluorescent protein, a cDNA encoding a nonfluorescent HaloTag protein or protein fusion is introduced
into cells either by transient transformation or generation of
stably transformed cell lines. By contrast to commonly used
fluorescent proteins, HaloTag proteins are expressed as
monomers. These cells are briefly incubated with an
appropriate HaloTag ligand which readily crosses the cell
* To whom correspondence should be addressed. E-mail: [email protected]
Abbreviations: CaMV, cauliflower mosaic virus; cLSM, confocal laser scanning microscopy; GFP, green fluorescent protein; mRFP, monomeric red
fluorescent protein; PEG, polyethylene glycol.
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
2986 Lang et al.
membrane. The HaloTag ligand harbours a reactive linker
that covalently binds to the HaloTag protein and a flexible
reporter group that can be a fluorophore. Unbound ligand is
washed out and fluorescence can be detected in living
or fixed cells (Los et al., 2005). Since the HaloTag protein is of prokaryotic origin, endogenous activities are
not detectable in mammalian cells. The covalent bond is
highly specific and essentially irreversible, yielding a complex that is stable even under denaturing conditions
(Technical Manual; Promega, 2005).
Like other fluorescent proteins, HaloTag protein can be
fused in-frame with any protein or peptide sequence of
interest in N- and C-terminal orientation without disturbing
their function, so that physiological processes including
cellular and subcellular activities, protein trafficking, and
protein interactions can be studied in vivo and noninvasively over longer time periods (Los et al., 2005). As
shown for animal systems, cells expressing the HaloTag
protein and labelled with the HaloTag TMR (tetramethyl
rhodamine which is red with an excitation peak of 555 nm
and a fluorescence emission peak of 585 nm) ligand, the
diAcFAM (diacetyl derivative of fluorescein which is green:
494Ex/526Em) ligand, or the coumarin (blue: 353Ex/
434Em) ligand are brightly fluorescent. By contrast, cells
that do not express the HaloTag protein show no detectable
fluorescence under the same labelling and imaging conditions. The HaloTag ligands have no toxicity or morphological side-effects in the cell lines tested (Los et al.,
2005). Besides fluorescence labelling, HaloTag technology gives other additional functionalities with a ligand
containing biotin which is suitable for use as an affinity tag
to capture the protein of interest (Los et al., 2005).
In this paper, the use of the HaloTag technology was
evaluated for localization experiments in plant cells. Expression of the HaloTag protein was visualized in tobacco
and poplar cells followed by staining with the HaloTag
TMR and diAcFAM ligands. HaloTag technology was
found to be a flexible system generating efficient fluorescence in different cell types of plants.
Materials and methods
Plasmid construction and purification
All restriction endonuclease and ligase reactions were performed
using the buffer conditions recommended by their respective
manufacturers using standard techniques (Sambrook et al., 1989).
The cDNA of HaloTag was amplified from pHT2-vector (GenBank
Accession number AY773970, kindly provided by Promega) using
Taq-DNA polymerase (Peqlab, Erlangen, Germany), with the following primer set:
forward primer 59-tcg gat ccA TGg gat cag aaa tcg gta c-39
reverse primer 59-tag cat gct aTT Agc cgg cca gcc cgg-39.
The PCR product was cloned into pGEMTeasy (Promega) and
sequenced. After cutting with BamHI and SphI, the resulting fragment
(902 bp) was transferred into the pFF19-vector (Timmermans et al.,
1990) to create pFF19-HT. This vector harbours the cauliflower
mosaic virus (CaMV)-35S promoter with double enhancer and the
poly(A) sequence from CaMV-35S. Plasmid-DNA was prepared with
Qiagen Plasmid Midi-Kit (Qiagen, Hilden, Germany).
Co-transformation of either pFF-GFP-PTS1 (Nowak et al., 2004)
or pGreen0229:MPmRFP (Hellens et al., 2000), kindly provided
by Dr S Chapman (SCRI, Norwich, UK), was used to distinguish
between transformed and non-transformed cells.
Plant material
Experiments were performed with in vitro-grown cultures of
Nicotiana plumbaginifolia and Populus tremula3Populus alba (No.
7171-B4; Institut de la Recherche Agronomique, INRA, France)
grown on modified MS-based medium (Murashige and Skoog, 1962;
Lloyd and McCown, 1980). The soil-grown plants of N. tabacum and
N. benthamiana were cultivated in controlled environment chambers
(Hereaus-Vötsch, HPS 1500, Balingen, Germany) with a 14 h light
(300 lE mÿ2 sÿ1)/10 h dark period and relative humidity of 80%.
Isolation and transformation of protoplasts
In vitro shoot cultures of Nicotiana plumbaginifolia were used for
protoplast isolation. Plants were maintained on MS medium (Murashige
and Skoog, 1962) without growth regulators at 23 8C and 25 lE mÿ2
sÿ1 under a 16 h light/8 h dark regime. Mesophyll protoplasts were
isolated from 2–3-month-old plants after overnight digestion of
leaves in 0.6% w/v Onozuka Cellulase R10 (Duchefa, Haarlem, The
Netherlands) and 0.2% w/v Macerocyme (Duchefa) dissolved in T0medium (Crepy et al., 1982) but omitting Tween. For transformation,
13106 protoplasts were resuspended per 1 ml MaMg solution
(Negrutiu et al., 1987). Aliquots of 500 ll were incubated at 45 8C
for 5 min, cooled on ice for 30 s followed by addition of 20 ll plasmid
DNA (1 lg llÿ1) and 520 ll polyethylene glycol (PEG) solution
containing 0.1 M Ca(NO3)2, 0.4 M mannitol, and 30% PEG 4000
(Merck, Darmstadt, Germany). After 20 min incubation at room
temperature, the PEG solution was removed with washing solution
W5 (Negrutiu et al., 1987) by centrifugation at 80 g for 5 min.
Protoplasts were cultured at a density of 13105 mlÿ1 in liquid T0
medium supplemented with 5 mM glutamine in the dark at 25 8C
using 6-cm-diameter Petri dishes.
Transformation of leaves using biolistics
Fully developed leaves of tobacco as well as poplar plants grown in
soil or in vitro were harvested; leaf discs (3 cm in diameter) were cut
with a metal punch and placed upside down on water-soaked filter
paper in Petri dishes. Coating of gold particles with plasmid DNA
was carried out as described earlier (Koprek et al., 1996). The
transformation was performed with the Particle Delivery System
PDS-1000 (Bio-Rad, Munich, Germany) using pressures of 350 and
700 psi and a distance of 45 and 75 mm between the macrocarrier
and the target tissue. Plant material was incubated in low light conditions at 25 8C. First analyses were done 24 h post-bombardment.
Staining of plant material with Calcoflour White, TMR and
diAcFAM ligands
After checking the transformation efficiency using GFP or monomeric red fluorescent protein (mRFP), the plant material was stained
as follows: transformed protoplasts or protoplast-derived cultures 2–
14 d after transformation were collected by centrifugation at 80 g for
5 min. The pellet was resuspended in 200 ll of W5 and 200 ll of
2-fold concentrated ligand solution dissolved in W5 was added
with a final concentration of 0.2, 1.0, and 5.0 lM. After incubation
for 15–60 min in the dark, the staining solution was carefully removed by washing twice with 10 ml W5, and protoplasts were
resuspended in 200 ll W5. The presence of a new cell wall was
HaloTagTM: a new plant reporter gene
2987
determined using the fluorescent brightener Calcofluor White
(Sigma, Deisenhofen, Germany) as described by Nagata and Takebe
(1970). One volume of 0.1% w/v Calcofluor White dissolved in W5
was added to the samples and analyses were carried out with cLSM.
Selected leaf areas or stripped lower epidermis of transformed
leaves were incubated for 30 min in 1.0 lM ligand solution diluted in
water. Careful washing for at least 4 h up to overnight in water was
necessary to reduce unspecific background.
Microscopic detection
Reporter gene expression was visualized with the confocal laser
scanning microscope cLSM-510META (Carl Zeiss, Göttingen,
Germany) using a sample of transformed protoplasts, selected leaf
areas, or the stripped lower epidermis of leaves. The specimens were
examined either using the Plan-Neofluar 103/0.3 or the C-Apochromat
403/1.2 water-immersion objectives. For confocal microscopy, the
argon laser (488 nm line for GFP, diAcFAM, and chlorophyll autofluorescence) and the helium laser (543 nm) for mRFP and TMR
were used. The emitted light was detected as follows: with the bandpass filter GFP (505–530 nm), diAcFAM (505–550 nm), TMR (560–
615 nm), and mRFP (560–615 nm), and with the META-channel
the chlorophyll autofluorescence. Additionally, a UV-laser (364 nm)
was used to detect Calcofluor White; primary beam splitting mirrors
UV/488/543/633 nm, emitted light was detected with a band pass
filter 385–470 nm using the Multitracking mode. If necessary, the
bright field of samples was taken with the transmitted light photomultiplier, and the lambda mode was used to examine the spectral
signature of the fluorochromes.
Results and discussion
Fig. 1. HaloTag transformation vector pFF19-HT: pBluescript-based
high copy plasmid carrying the cDNA of HaloTag protein (HT) under the
control of the CaMV-35S promoter with double enhancer and the
poly(A) sequence from CaMV-35S.
transformation marker pFF-GFP-PTS1 facilitates transfer of
GFP directly into peroxisomes. The other transformation
marker pGreen0229:MPmRFP is described as a plasmodesmata marker (S Chapman, personal communication). Thus
in both cases, an accumulation of the fluorochromes GFP
and mRFP was expected in distinct cell compartments,
whereas the HaloTag protein should be distributed over
the cytoplasm and the nucleus because of the small size of
the protein (33 kDa).
Vector construction and transformation
The cDNA of HaloTagTM protein was transferred from the
pHT2-vector (Promega) into a plant-specific expression
system via PCR-based cloning. Intermediate cloning steps
were performed in the pGEMTeasy-vector (Promega).
Expression of the protein was driven by the strong
CaMV-35S promoter with double enhancer in the pFF
vector (Timmermans et al., 1990), a pUC-based high copy
plasmid for direct DNA-transfer techniques. The final pFFHT plasmid is shown in Fig. 1.
Gene transfer experiments were started using PEGmediated protoplast transformation. Protoplasts, as plant
cells surrounded only by a plasmamembrane, were chosen
to investigate the possible negative effect of the plant
cell wall for staining. Cell walls are known to adsorb the
fluorochromes and could therefore disturb or inhibit the
colouring procedure. Subsequently, biolistics was used for
transformation of intact plant tissues.
In both approaches, protoplast transformation and biolistics, a second plasmid was co-transformed in order to
distinguish between transformed and non-transformed plant
cells. For this purpose, the constructs pFF-GFP-PTS1
(Nowak et al., 2004) or pGreen0229:MPmRFP were used.
The targeting signal PTS1 is a C-terminal sequence (SKL,
SNL, or other variants; Gould et al., 1989; Mullen et al.,
1997) known as signal targeting exclusively for peroxisomes (for a review, see Sparkes and Baker, 2002). Thus the
Detection of transiently expressed fluorescent
proteins in protoplasts
Protoplasts have the advantage being comparable to animal
cells due to the removal of the plant cell wall. Two days
after PEG-mediated transformation of N. plumbaginifolia
protoplasts, the transformation efficiency was checked
using GFP and mRFP fluorescence, respectively. Incubation with TMR or diAcFAM ligand was performed for 15,
30, or 60 min in a final concentration of 0.2, 1.0, and 5.0
lM ligand solution. After two washing cycles the cells were
immediately subjected to fluorescence microscopy.
The protoplasts showed a clear GFP, mRFP, TMR, and
diAcFAM fluorescence (Fig. 2). It was found that staining
for 30 min with 1.0 and 5.0 lM ligand solution gave
brilliant signals, whereas 0.2 lM was not sufficient to
colour the material. Two protoplasts stained with 1.0 lM
TMR are shown in Fig. 2a–e, one of them (the lower one)
has a clear GFP-fluorescence. As pointed out before, this
GFP is nearly exclusively localized in peroxisomes mediated by the PTS1 sequence (Nowak et al., 2004). Only this
protoplast had a strong TMR fluorescence (in the fourth
channel of the cLSM) which is exclusively localized in the
cytoplasm because the HaloTag protein possesses no
targeting signal.
As a next step, the lambda mode of the cLSM510META (Zeiss) was used to prove the specificity of TMR
2988 Lang et al.
Fig. 2. Images of tobacco cells with transient expression of HaloTag protein and GFP/mRFP. Protoplasts and protoplast-derived cells are visualized in
the transmitted light channel (a, f, l), GFP (b, g, m), chlorophyll (c, h, n), TMR (d, i, o), and Calcofluor White fluorescence (j, p) and in the merged picture
(e, k, q). Only the lower protoplast in (a–e) which shows GFP fluorescence in the peroxisomes 2 d after transformation (b) also has TMR fluorescence in
HaloTagTM: a new plant reporter gene
fluorescence. The META-detector of the cLSM-510,
a combination of an optical grating in line with a 32channel photomultiplier array, can acquire simultaneously
several emission signals with complete separation of their
wavelengths. This allowed a reference spectrum (emission
fingerprint) of the colours showing an emission maximum
at 585 nm for TMR (Fig. 2r) and 522 nm for diAcFAM to
be taken (Fig. 2s). No unspecific labelling was detectable
with the TMR ligand, either from unknown cell compounds or by cell walls. Only for diAcFAM was an
unspecific labelling of the vacuole and of a few chloroplasts found in some cases. The diAcFAM ligand is
based on a non-fluorescent diacetyl derivative of fluorescein which is converted to a brightly fluorescent fluorophore upon cleavage of the diacetyl groups by cellular
esterases, known also from determination of cellular
viability assays (Amano et al., 2003). Fluorescein itself
is no longer membrane-permeable which could be the
reason for the unspecific staining found. No cross-talk
was observed between the channels: TMR and diAcFAM
ligands, red autofluorescence of chloroplasts, mRFP, and
the GFP of peroxisomes were clearly separated from
each other.
The following experiments were designed to find out if
the newly developing cell wall of transformed protoplasts
can interfere with the colouring process. Therefore protoplast-derived cultures (4 and 14-d-old) were incubated
with the TMR and diAcFAM-ligand solutions and the cell
wall stained in parallel with Calcofluor White. Figure 2j
and p illustrates cell wall staining. The two protoplasts in
Fig. 2f–k started to develop a cell wall and therefore look
more oval-shaped. Figure 2l–q shows the first division of
a protoplast-derived cell with a compact cell wall. In both
cases the transformed cells, which express GFP transiently
in the peroxisomes, also have an intensive TMR fluorescence in the cytoplasm. These results demonstrate that the
staining of transiently expressed HaloTag protein in intact
plant cells is not hampered by the plant cell wall.
Detection of transiently expressed fluorescent
proteins in intact plant cells
Biolistic transformation was used to transform intact plant
cells. Leaf material of two tobacco species (Nicotiana
tabacum cv. Gatersleben and N. benthamiana) and from
poplar (Populus tremula3Populus alba) was tested for
possible application in herbaceous plants and trees, shooting routinely into the upper epidermis. Two days after
bombardment, the leaf discs were inspected and GFP/
mRFP-expressing areas were cut out for subsequent staining. Based on the results of the protoplast experiments,
2989
only the 1.0 lM ligand solution and staining for 30 min
were used, and the focus was mainly on the destaining
procedure.
Immediately after staining and destaining, a high background of TMR and diAcFAM fluorescence was found
(data not shown), and even 4 h destaining in water did not
completely eliminate this background fluorescence. Therefore, longer destaining was essential. Overnight incubation
in water gave the pictures shown in Fig. 3. A GFP- or
mRFP-expressing cell gave a strong TMR or diAcFAM
fluorescence, respectively. There was nearly no background
in the surrounding cells as shown in the low magnification
picture for diAcFAM (Fig. 3j) and TMR (Fig. 3k). The
epidermal cell (Fig. 3a–e for N. tabacum and Fig. 3l, m for
N. benthamiana) and the guard cell of N. tabacum (Fig. 3f–
i) showed an intensive TMR fluorescence exclusively in
the cytoplasm. Since the size exclusion limit of the nucleus
is in the range of 40–60 kDa (Shiota et al., 1999), and the
protein used has a molecular weight of 33 kDa, staining
of the nucleus was also detected (Fig. 3e, i, j). In Fig. 3f–i,
only the guard cell with the GFP fluorescence (=transformation marker; Fig. 3f) also had TMR fluorescence
(Fig. 3h); the second cell of the stomatal apparatus was not
transformed during bombardment and hence served as
a negative control. The reference spectra of the colours
detected with the META detector of the cLSM-510 showed
an emission maximum for TMR and diAcFAM in the
range found for the protoplasts (Fig. 2t, u). There were no
differences when using intact leaf areas or stripped lower
epidermis of leaves during the observation on the cLSM,
except that the transmitted light channel gave a more
brilliant picture in the case of the mono-cell layer of the
epidermis.
In addition to tobacco, poplar was also tested as the
model for woody plants (Taylor, 2002) and leaf material
from in vitro and hydroponically grown plants was transformed. The results are summarized in Fig. 3n and o. In
some cases, there were problems with destaining of the hydroponically grown leaf material because of the big masses
of hairs that created obstacles either for the penetration of
gold particles into epidermal cells or for destaining.
Benefits of the HaloTag protein for plant investigations
The HaloTagTM-technology as a new detection system for
rapid, site-specific labelling of proteins was first established
in mammalian cells. The technology is based on the
formation of a covalent bond between the HaloTag protein,
which is transiently or stably expressed in cells, and a
synthetic ligand which is used in a staining procedure
prior to visualization. These ligands carry a variety of
the cytoplasm (d); the upper protoplast serves as a negative control. Four days after transformation (f–k), the protoplasts developed a new cell wall (j);
here also only the cell with GFP fluorescence (g) has a clear TMR signal (i). Both daughter cells of a dividing protoplast-derived cell have GFP
fluorescence (m), the thick cell wall (p) does not disturb the staining with TMR (o). In (r–u), the spectral signatures taken by the META channel of the
different fluorochromes are presented in protoplasts of N. plumbaginifolia (r, s) and epidermal cells of N. tabacum (t, u). Scale bars=10 lm.
2990 Lang et al.
Fig. 3. Images of plant cells with transient expression of HaloTag protein after biolistic transformation. Epidermal (a–e, j) and guard cells (f–i) of N.
tabacum are visualized in the transmitted light channel (a), GFP (b, f), chlorophyll (c, g), and TMR fluorescence (d, h) and, in the merged picture, with
TMR (e, i), and diAcFAM fluorescence (j). Nicotiana benthamiana guard cells expressing TMR and GFP fluorescence are shown in a low magnification
overview (k), a section with only the TMR signal (l), and in the merged picture (m). TMR fluorescence in Populus tremula3Populus alba is presented
in (n) and in the merged picture with GFP and chlorophyll fluorescence (o). Scale bars=20 lm.
HaloTagTM: a new plant reporter gene
functionalities, including fluorescent labels, affinity tags,
and attachments to a solid phase (Los et al., 2005). The
efficiency of transformation and expression of the HaloTag
protein followed by staining ligands in plant cells was
studied. A high transformation and labelling efficiency
under the conditions used (strong promoter and protoplast
transformation or biolistics) were found. Depending on the
experiments, 2–30% of the protoplasts or in the biolistics
30–150 cells per bombarded leaf area showed transient
foreign gene expression. In all of the cells observed, the
co-transformation rate of the two plasmids used was in
the 100% range, which means that, in the case where GFP
fluorescence was found, the Halo-Tag could also be detected and vice versa.
The HaloTag-system is comparable to the well-established GFP system (Haseloff and Amos, 1995). It allows
fusion of any protein of interest to its N- or C-terminus. The
molecular weight of the protein (33 kDa) does not
significantly diverge from the size of GFP (27 kDa). As
with any fusion protein, fusing with HaloTag protein may
affect the functionality of the protein. However, the incorporation of an appropriate peptide linker between the
protein of interest and the HaloTag protein may help to
minimize this effect (Los et al., 2005). Since HaloTag
proteins are expressed as monomers and exhibit a compact protein structure, steric hindrance of genetic fusions is
strongly reduced. In terms of flexibility, the HaloTag
technology offers additional benefits in comparison to the
use of fluorescent proteins. Because of the open architecture of the technology which allows the use of different
ligand colours, one cloning procedure is sufficient. Thus, timeconsuming re-cloning work is reduced, which is important
for co-labelling experiments where two or more fluorescent
proteins need to be tested to overcome background issues.
To this end, Promega offers three different fluorescently
labelled ligands and two ligands containing biotin; additional ligands are under development (Truc N Bui, personal
communication). When analysed in mammalian cell lines,
the HaloTag TMR, coumarin, diAcFAM, and biotin ligands
are cell permeable and show no toxicity or morphological
side-effects at recommended labelling conditions in the cell
lines tested (Los et al., 2005). These results are consistent
with what was found using living plant cells. Moreover,
comparable to mammalian systems, plant material can be
fixed in 3% formaldehyde solution and mounted in Mowiol
4.88 (Hoechst Farbwerke, Frankfurt, Germany) (data not
shown). In addition to high flexibility, the use of HaloTagTM
Interchangeable Labeling Technology will help to reduce
toxicity issues due to overexpression of GFPs, as discussed
by Haseloff et al. (1997). These authors pointed out that
the accumulation of fluorescent proteins led to the generation of free radicals, especially H2O2, in cells due to the
formation of the chromophore by cyclization, dehydration,
and aerial oxidation which has not been reported in the
HaloTag-system. It was found that a slow decrease in the
2991
number and intensity of the stained cells, as known for
transient gene expression, could be monitored in the ‘long
term’ transient protoplast system for 14 d. But no dramatic
drop was seen as would have been expected for a toxic
substrate. For live cell imaging studies that require longterm microscopic observation, toxicity issues need to be
considered to establish proper experimental outcomes.
Acknowledgements
This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ha3107/3-1). We are grateful to students Michael
Schaller, Jana Tiefenau, and Yasemine Sömer for excellent technical
work during their practical course in our laboratory, and to Promega
(Dr Stephan Brockmann and Dr Truc N Bui) for providing the
pHT2-Vectorsystem, the colouring solution, and helpful discussion.
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