xCELLigence System Real-Time Cell Analyzer
Focus Application
Cellular Analysis
For life science research only.
Not for use in diagnostic procedures.
Introduction
The precise regulation of gene expression is a very important
feature of how organisms respond to environmental changes
and regulate cell proliferation, development, and even
programmed cell death. Gene expression starts with the
transcription of genomic DNA into messenger RNA, the
template for protein synthesis during ribosomal translation.
Eukaryotic cells use three different RNA polymerases, each
producing one of the main categories of cellular RNA:
Pol I transcribes rRNA (28 S, 18 S and 5.8 S rRNA), Pol II
transcribes mRNA, and Pol III transcribes the 5 S rRNA and
tRNA. Only Pol II transcripts are translated into proteins.
Pol II is also modified by enzymes influencing different
stages of transcription, processing of premature mRNAs in
a promoter-dependent manner. These modifications occur
in a domain at the carboxy-terminal part of the largest
subunit of Pol II, which has a very unique structure.
Twenty years after the first description of the RNA polymerase II C-terminal domain, its function remains unclear.
Pol II’s evolution in conjunction with chromatin and mRNA
processing, suggests it serves as an integrator of signalling
events for regulating gene expression at multiple levels
(Chapman et al. 2008). Across species, the C-terminal
domain (CTD) is highly conserved, indicating conserved
functions for nearly all its residues, forming a repetitive
structure of 52 repeats in humans (and 26 in yeast), with
the consensus sequence Y1S2P3T4S5P6S7 (Corden et al.
1985). This sequence is striking in the fact that every residue
Martin Heidemann
Institute for Clinical Molecular Biology and Tumour Genetics,
Helmholtz Center for Environmental Health, Center for
Integrated Protein Sciences (CiPSM), Marchioninistr. 25,
D-81377 Munich, Germany.
can be enzymatically modified. During the transcription
cycle, differential phosphorylation of CTD residues occurs
concomitant with promoter escape, orchestrating the
recruitment, activation, and displacement of different
factors involved in transcription control and mRNA
processing from the CTD at different points across a gene
(Chapman et al. 2007).
Results
Our group established a system allowing the production
and testing of entirely synthetic CTDs in vivo (Chapman
et al. 2004). While endogenous Pol II is strongly inhibited
by a-amanitin, the poison of the death cap mushroom
Amanita phalloides, our recombinant polymerases are
engineered with a point mutation (Asn793Asp) in the large
subunit conferring resistance to a-amanitin.
To determine optimal cell numbers for this experiment,
HEK 293 cells were titrated on a E-Plate 96 for use with
the xCELLigence System RTCA SP Instrument. Prior to
HEK 293 plating, background impedance was determined
using just 100 µl DMEM with 10% FCS. Subsequently,
20,000 to 1,250 HEK 293 cells were plated per well and the
Cell Index values (CI) were continuously monitored for
3 days (see Figure 1).
Issue 01
Featured Study:
Analysis of RNA Polymerase II Mutants
using the xCELLigence System
Cell Index
2
7 µl 6 µl 5 µl 4 µl 3 µl
lI
IIo
IIa
IIb
2.84
Po
l
3.26
rec. Pol II
8 µl c.
ro
nt
co
co
nt
ro
3.68
re
l
I D0
+5
4.10
FuGENE® HD/
2 µg DNA
anti-HA
(clone 3F10)
2.42
control
2.00
Lane 123
1.58
4 5678
9
Figure 3: Immunoblotting of HEK 293 cells transfected with HA-tagged Pol II.
FuGENE® HD Transfection Reagent titration reveals increasing Pol II expression 24 hours
after transfection (lane 4-9). Indicated are the hyperphosphorylated (IIo), the hypophosphorylated (IIa), and the CTD-lacking (IIb) isoforms of Pol II (Chapman et al. 2005).
1.16
0.74
0.32
-0.10
22.0 33.0 44.0 55.0 66.0 77.0
Time (in Hour)
20.000 HEK 293
10.000 HEK 293
5.000 HEK 293
2.500 HEK 293
1.250 HEK 293
medium control
Figure 1: Cell number titration of HEK 293 cells in E-Plates 96 of the xCELLigence
System. The Cell Index was continuously monitored for 72 hours in 30 min intervals.
To further investigate CTD function, we analyzed transiently
transfected HEK 293 cells, expressing recombinant wild-type
Pol II or Pol II lacking the entire CTD except repeat 52
(0+52). Cell proliferation was continuously monitored
throughout the experiment using the xCELLigence System
(ACEA), in order to record the cellular response to (i)
transfection and (ii) a-amanitin treatment. We plated 5000
HEK 293 cells in a final volume of 150 µl DMEM (10% FCS)
per well on an E-Plate 96. After 24 hours, cells were transfected with 0.1 µg plasmid DNA per 0.4 µl FuGENE® HD
Transfection Reagent (Roche). After an additional 24 hours,
cells were treated with a-amanitin to inhibit endogenous
Pol II and the CI was continuously monitored for 120 hours.
As shown using continuous recording with the xCELLigence
System, the optimized transfection protocol (8 µl FuGENE®
HD Transfection Reagent per 2 µg DNA) did not affect HEK
293 proliferation 40 hours after transfection (see Figure 4),
clearly avoiding potential nonspecific side effects.
1.20
1.07
0.94
0.81
0.68
transfection
11.0 Cell Index
0.0 0.55
0.42
0.29
0.16
0.03
-0.10
0.0 8.0 16.024.0 32.040.048.0 56.064.0
Time (in Hour)
HEK 293
HEK 293 + FuGENE ® HD
Figure 4: Cell proliferation assay of HEK 293 cells transfected with FuGENE® HD
Transfection Reagent. Proliferation of non-transfected HEK 293 cells is shown in green.
Wild-type cells treated with the optimized transfection protocol are depicted in red.
phase contrast
eGFP
merged
Figure 2: Optimization of HEK 293 transfection efficiency with FuGENE ® HD
Transfection Reagent. Transient expression of eGFP was monitored by phase contrast
microscopy 24 hours after transfection.
Transfection with FuGENE® HD Transfection Reagent
was optimized in standard 96 well cell culture dishes
(see Figure 2), according to the protocol for optimizing
transfection of adherent cell lines (FuGENE® HD Transfection
Reagent Application Note No. 3). Transfection efficiency
was monitored by immunoblotting, revealing highest
recombinant Pol II expression with 8 µl FuGENE® HD
Transfection Reagent per 2 µg plasmid DNA (see Figure 3).
Recombinant Pol II protein was detected using the Anti-HA
High Affinity antibody (clone 3F10) from Roche.
As shown in Figure 5, non-transfected HEK 293 cells
proliferate continuously for 90 hours after plating. After
90 hours of incubation the CI values rapidly decrease,
most likely due to the consumption of the cell culture
medium, which causes a detachment of the cells from
the cell culture dish and eventually cell death. Treatment
of non-transfected HEK 293 cells with a-amanitin causes
a rapid drop in the CI values already 24 hours after
compound treatment, indicating large-scale cell death
due to the specific inhibition of Pol II.
2.9
2.6
2.3
a-amanitin
Cell Index
2.0
1.7
1.4
1.1
0.8
0.5
Conclusion
In conclusion, the new xCELLigence System technology
allows for the first time the monitoring of cell proliferation
and cell death throughout the entire experiment, constantly
recording cellular effects during cell plating, transfection,
and compound treatment. This non-invasive and continuous monitoring of cells provides a far more detailed picture
into the whole experimental process, revealing both specific
and off-target effects that may have been missed using
conventional endpoint assays.
0.2
-0.1
0.015.0 30.0 45.060.075.0 90.0106.0
Time (in Hour)
HEK 293
HEK 293 + a-amanitin
Figure 5: Cell proliferation assay of HEK 293 cells treated with the Pol II inhibitor
-amanitin. Proliferation of non-transfected HEK 293 cells is shown in green. HEK 293 cells
treated with the specific Pol II inhibitor a-amanitin are depicted in red.
Functional characterization of the Pol II CTD domain was
performed by monitoring proliferation of HEK293 cells
transfected with recombinant Pol II mutants (see Figure 6).
HEK 293 cells with the recombinant wild-type Pol II (red)
continued to proliferate after inhibition of endogenous
Pol II by a-amanitin. This result demonstrates that the
reconstitution of Pol II expression with the a-amanitinresistant Pol II mutant compensates the loss of endogenous
Pol II function, thus rescuing HEK 293 cells from cell death.
Cells transfected with the recombinant Pol II mutant lacking
the CTD die abruptly after the addition of a-amanitin.
As these transfected HEK 293 cells die even faster than the
non-transfected negative control, it can be concluded that
the CTD-deficient mutant Pol II0+52 has a dominant
negative effect aggravating the toxic effect of a-amanitin.
2.9
a-amanitin
1.7
1.4
1.1
0.8
transfection
Normalized Cell Index
2.0
2.Chapman RD, Heidemann M, Albert TK, Mailhammer R,
Flatley A, Meisterernst M, Kremmer E, Eick D. (2007).
“Transcribing RNA polymerase II is phosphorylated at
CTD ­residue serine-7.” Science 318(5857): 1780-2.
3.Chapman RD, Heidemann M, Hintermair C, Eick D.
(2008). “Molecular evolution of the RNA polymerase II
CTD.” Trends in Genetics Vol 24 No.6.
4.Chapman RD, Palancade B, Lang A, Bensaude O, Eick D.
(2004). “The last CTD repeat of the mammalian RNA
­polymerase II large subunit is important for its stability.”
Nucleic Acids Res 32, 35-44.
5.Corden JL, Cadena DL, Ahearn JM Jr, Dahmus ME.
(1985). “A unique structure at the carboxyl terminus of
the ­largest ­subunit of eukaryotic RNA polymerase II.”
Proc Natl Acad Sci U S A 82(23): 7934-8.
6.FuGENE® HD Transfection Reagent, Application Note
No. 3 (November 2006). Protocol for optimizing
­t ransfection of ­adherent cell lines.
2.6
2.3
References
1.Chapman RD, Conrad M, Eick D. (2005).
“Role of the mammalian RNA polymerase II C-terminal
domain (CTD) nonconsensus repeats in CTD stability
and cell proliferation.” Mol Cell Biol. Sep;25(17).
0.5
0.2
-0.1
0.0 16.032.0 48.064.080.0 96.0112.0128.0
Time (in Hour)
HEK 293 + rec. Pol II + a-amanitin
HEK 293 + rec. Pol IID0+52 + a-amanitin
Figure 6: Cell proliferation assay of HEK 293 cells transfected with recombinant
Pol II. Proliferation of HEK 293 cells transfected with the recombinant full-length Pol II
is shown in green. HEK 293 cells transfected with the CTD-deficient Pol II mutant are
depicted in red.
Ordering Information
Product
Cat. No.
Pack Size
xCELLigence RTCA DP Instrument
RTCA DP Analyzer
RTCA Control Unit
00380601050
05469759001
05454417001
1 Bundled Package
1 Instrument
1 Notebook PC
xCELLigence RTCA SP Instrument
RTCA Analyzer
RTCA SP Station
RTCA Control Unit
00380601030
05228972001
05229057001
05454417001
1 Bundled Package
1 Instrument
1 Instrument
1 Notebook PC
xCELLigence RTCA MP Instrument
RTCA Analyzer
RTCA MP Station
RTCA Control Unit
00380601040
05228972001
05331625001
05454417001
1 Bundled Package
1 Instrument
1 Instrument
1 Notebook PC
E-Plate 16
05469830001
05469813001
06324738001
06324746001
06465382001
6 Plates
6 x 6 Plates
6 Plates
6 x 6 Plates
1 x 6 Devices (6 16-Well Inserts)
CIM-Plate 16
05665817001
05665825001
6 Plates
6 x 6 Plates
E-Plate 96
05232368001
05232376001
06472451001
06472460001
06465412001
06465455001
6 Plates
6 x 6 Plates
6 Plates
6 x 6 Plates
1 x 6 Devices (36 16-Well Inserts)
6 Units (6 Receiver Plates + 6 Lids)
E-Plate VIEW 16
E-Plate Insert 16
E-Plate VIEW 96
E-Plate Insert 96
E-Plate Insert 96 Accessories
For life science research only.
Not for use in diagnostic procedures.
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