A previously functional tetracycline-regulated transactivator fails to target gene expression to the bone
BMC Research Notes
A previously functional tetracycline-regulated transactivator fails to target gene expression to the bone
Eva Schmidt 0
Maria Eriksson 0
0 Department of Biosciences and Nutrition, Center for Biosciences , Karolinska Institutet , Karolinska University Hospital, Huddinge, Novum , SE-14183 Stockholm , Sweden
Background: The tetracycline-controlled transactivator system is a powerful tool to control gene expression in vitro and to generate consistent and conditional transgenic in vivo model organisms. It has been widely used to study gene function and to explore pathological mechanisms involved in human diseases. The system permits the regulation of the expression of a target gene, both temporally and quantitatively, by the application of tetracycline or its derivative, doxycycline. In addition, it offers the possibility to restrict gene expression in a spatial fashion by utilizing tissue-specific promoters to drive the transactivator. Findings: In this study, we report our problems using a reverse tetracycline-regulated transactivator (rtTA) in a transgenic mouse model system for the bone-specific expression of the Hutchinson-Gilford progeria syndrome mutation. Even though prior studies have been successful utilizing the same rtTA, expression analysis of the transactivator revealed insufficient activity for regulating the transgene expression in our system. The absence of transactivator could not be ascribed to differences in genetic background because mice in a mixed genetic background and in congenic mouse lines showed similar results. Conclusions: The purpose of this study is to report our negative experience with previously functional transactivator mice, to raise caution in the use of tet-based transgenic mouse lines and to reinforce the need for controls to ensure the stable functionality of generated tetracycline-controlled transactivators over time.
The tetracycline-inducible system (tet-ON/OFF) is a
binary transgenic system that enables spatial and
temporal regulation of gene expression. It consists of an
inducible transcriptional activator (tTA or rtTA) and a
tetracycline-responsive promoter element (TRE element,
tetop) that controls the transcription of a target gene
sequence [1,2]. By placing the sequence of the tTA or
rtTA downstream of a tissue-specific promoter, it
enables the distinct spatial regulation of expression of a
gene of interest inside the mammalian organism .
Furthermore, the target gene expression can be
regulated quantitatively by exposing the system to
tetracycline or its derivative, doxycycline. Temporal control
might be of special interest to overcome toxic or lethal
reactions from gene products expressed during early
development but that one might want to study at later
time points . In the tet-OFF system, the
tetracyclinecontrolled activator (tTA) is only active in the absence
of doxycycline and binds to the TRE element, which
controls the target transgene . In contrast, the reverse
tetracycline-controlled activator (rtTA), utilized in the
tet-ON system, requires doxycycline to be activated and
to bind to the TRE element .
Numerous transgenic mouse models have been
developed utilizing this system to control gene activity in a
broad range of biological systems , and more than
100 transactivator (tTA or rtTA) and responsive (pTRE)
strains have been published . Our laboratory has
generated transgenic mice with different minigenes of
wildtype and mutant human lamin A, under the control of
the TRE element (tetop), to study the molecular
mechanisms underlying Hutchinson-Gilford progeria
syndrome (HGPS) [9-11]. The disease is caused by a de
novo point mutation in exon 11 of the LMNA gene
(1824C > T, G608G). The mutation results in the
activation of a cryptic splice site and an abnormally processed
prelamin A protein named progerin [12,13]. HGPS is a
rare, segmental progeroid syndrome where the most
striking phenotypes affect the skin, the skeletal system
and the cardiovascular system. Especially because of the
severity of the symptoms of HGPS, the tet system would
be useful for studying this disease in transgenic animals.
Targeted expression of the lamin A minigene carrying
the HGPS mutation (tetop-LAG608G) to the skin in mice,
using the keratin 5 transactivator (K5tTA) , leads to
epidermal disease with similar clinical features as have
been reported in HGPS patients . To study the
skeletal abnormalities in HGPS, we decided to utilize the
previously published collagen type 1a1-rtTA (a1p-rtTA)
system to create a bone-specific expression model of the
HGPS mutation. Even though the transactivator mice
had previously been reported to exhibit bone-specific
Smad1C expression in the presence of doxycycline ,
when we crossed two founder lines expressing lamin A
minigenes, tetop-LAG608G VF1-07 and tetop-LAWT
SF104, to generate tetop-LAG608G VF1-07; a1p-rtTA and
tetop-LAWT SF1-04; a1p-rtTA mice, neither show
detectable expression of human lamin A proteins in
Materials and methods
Genetically modified mice
a1p-rtTA promoter mice were maintained in mice on
C57BL/6J and backcrossed to FVB/NCrl for ten
generations to generate FVB/N.Cg-Tg(a1p-rtTA). Transgenic
tetop-LAG608G mice, line VF1-07, generated on FVB/
NCrl , were backcrossed to C57BL/6J for ten
generations to generate B6.Cg-Tg(tetop-LAG608G,-EGFP)
VF1-07. Transgenic tetop-LAWT mice, line SF1-04,
generated on FVB/NCrl , were maintained on FVB/
NCrl. Animals were housed in a 12-hour light/dark
cycle at 20-22°C and 55-65% air humidity in a
pathogen-free animal facility at the Karolinska University
Hospital. The mice were fed irradiated mouse pellets
RM3 (Scanbur BK AB). To target HGPS transgenic
expression to bone tissue, the mice were intercrossed
with a1p-rtTA transgenic mice. To induce transgenic
expression, doxycycline (200 μg/ml) in drinking water
containing 2.5% sucrose was given during intercross
breedings or from postnatal day 0. Offspring from
FVB/N-Tg(tetop-LAG608G,-EGFP)VF107 and B6.Cg-Tg(a1p-rtTA), and
FVB/N-Tg(tetopLAWT,-EGFP)SF1-04 and B6.Cg-Tg(a1p-rtTA), were
regarded as 50:50 mixed background (FVB/N;C57BL6).
Offspring from intercross breedings,
B6.Cg-Tg(tetopLAG608G,-EGFP)VF1-07, and B6.Cg-Tg(a1p-rtTA), and
FVB/N-Tg(tetop-LAG608G,-EGFP)VF1-07 and FVB/N.
Cg-Tg(a1p-rtTA) were regarded as pure background.
Offspring from intercrosses between
FVB/N-Tg(tetopLAG608G,-EGFP)VF1-07 and additional transactivator
lines, B6.Cg-Tg(Sp7-tTA) and B6.Cg-Tg(SM22-rtTA),
were used as controls. All mice were genotyped using
published protocols [10,15-17]. Mice were born at the
expected Mendelian ratios. The animal studies were
approved by the Stockholm South Ethical review board,
Dnr. S148-03, S141-06 and S107-09.
RNA and protein extraction
Animals were sacrificed with an overdose of isoflurane
(Baxter) at postnatal week 5, 20 or 46, and the long
bones, tibia and femur were dissected and the bone
marrow removed. The bone tissue was crushed with the
Bessmann tissue homogenizer. Bone pieces were
transferred to Lysing Matrix D, and Fastprep 220A
(Qbiogene) was used twice to further homogenize at 6 m s-1
for 20 seconds. Samples were incubated on ice for 10
minutes between the runs. RNA was extracted using
TRIZOL (Invitrogen), and protein was extracted using 8
M Urea/RIPA buffer (containing proteinase inhibitor,
Roche). Abdominal aorta was collected from adult
tetop-LAG608G;SM22-rtTA transactivator mice , and
mRNA was extracted according to the manufacturer’s
instructions (Micro Fasttrack™ 2.0 mRNA isolation kit,
Invitrogen). To homogenize aorta, Lysing Matrix D and
Fastprep 220A was used at 6 m s-1 for 40 seconds.
RT-PCR and western blot analysis
cDNA was synthesized using random primers according
to the instructions for the SuperScript™First-Strand
Synthesis System (Invitrogen). RT-PCR for human lamin
A and laminAdel150 were performed as previously
described . The RT-PCR protocol published by Liu et
al.  was used to examine the expression of rtTA. PCR
amplification of cDNA using primers for b-actin
(5’CCTAGGCACCAGGGTGTGAT-3’ and 5’-CCATGTCG
TCCCAGTTGGTAA-3’) was performed on all samples
as a control. Western blot was performed in accordance
with previously published procedures . Primary
antibodies used for western blot were mouse monoclonal
anti-human lamin A+C (mab3211, Chemicon) and
mouse monoclonal anti-b-actin (A5441, Sigma).
Primary osteoblast cultures
The procedure for isolation and culture of primary bone
cells from adult mouse calvariae was modified from the
protocol published by Bakker and Klein-Nulend . In
brief, animals at the age of 5 or 13 weeks were
euthanized with an overdose of isoflurane (Baxter). The
calvaria was collected and immediately rinsed in phosphate
buffered saline (PBS) supplemented with antibiotics (1x
AB-AM, Gibco). Bone pieces were cut into smaller
fragments and incubated in 2 ml collagenase solution (2
mg/ml Collagenase II, Worthington Biochemical in
alpha-MEM M8042, Sigma) and 2 ml 1x Trypsin-EDTA
(Gibco) at 37°C on a shaking rotarod. Sequential
digestion was performed as described by Bakker and
KleinNulend . The calvaria pieces were placed in 60-mm
culture dishes (Sarstedt) and cultured in 5 ml osteoblast
culture medium (alpha-MEM M8042 (Sigma) with 1x
AB-AM (Gibco) and 15% heat-inactivated fetal bovine
serum (SaveenWerner). The medium was changed every
third day until the cell cultures were confluent. Cells
were collected using Trypsin-EDTA (Gibco) and a cell
Osteoblasts (1 × 103) were washed and resuspended in
200 μl PBS. Cells were spun on glass slides (SuperFrost
Plus, Menzel) using the Shandon Cytospin 4 machine
with Shandon filter cards (Thermo) at medium
acceleration rate and 500 rpm for 15 minutes. Cells were
allowed to dry at RT before they were stored at -8°C.
Osteoblasts were fixed for 10 minutes in 4%
paraformaldehyde at room temperature and permeabilized for 5
minutes using 1% NP-40 in PBS (Pierce). The cells were
blocked using 5% serum/0.1% Birj (Pierce) in PBS for 30
minutes  and incubated with mouse monoclonal
anti-human lamin A+C antibody (mab3211, Chemicon)
overnight at 4°C in a 1:5 dilution of blocking solution.
On the following day, the cells were washed and
incubated with a secondary antibody, anti-mouse Alexa
Fluor 594 (1:1000 dilution, Invitrogen). A MIST tray
was used for all incubation steps. Cells were mounted
using Vectashield mounting media (H-1200, Vectorlab)
containing DAPI (4’,6-diamidino-2-phenylindole).
Results and discussion
In this study we have utilized the previously published
a1p-rtTA transgenic mice in combination with a
transgenic mouse line carrying a minigene of human lamin A
with the HGPS point mutation, tetop-LAG608G, to create
a bone-specific expression model for the disease. A
second transgenic mouse line, with only the human
wildtype lamin A sequence, tetop-LAwt, was used as a
control. To circumvent possible problems with genetic
background, the congenic strain
FVB/N.Cg-Tg(a1prtTA) was formed by backcrossing a1p-rtTA for ten
generations to FVB/NCrl. For the same purpose,
transfer-breeding for tetop-LAG608G, line VF1-07, generated
on FVB/NCrl, was performed to form congenic strain
B6.Cg-Tg(tetop-LAG608G,-EGFP)VF1-07. This breeding
protocol enabled the analysis of transgenes on both
mixed and pure genetic backgrounds.
To analyze the expression of the transgene and make
sure that the transactivator was present and able to
activate the expression of human lamin A in bone, we
performed RT-PCR experiments. RT-PCR for human lamin
A and lamin Adel150 only showed scant amplification
products after 35 cycles of PCR in
tetop-LAG608G+;a1prtTA+ and tetop-LAwt+;a1p-rtTA+ (Figure 1 and data
not shown). Scant amplification products were also
present in samples that did not have the transactivator,
tetop-LAwt+;a1p-rtTA(data not shown). This result indicated a potential
leakiness of the system and is a problem that has been seen
by others using this system [19-21]. However, the very
low expression could be an effect of inappropriate
transcription of the transgene, which might have been due
to the fact that the lamin A minigene integration site
was active in bone tissue, or an effect of low activity of
the transactivator. To directly assess if the transactivator
was present in bone tissue, we performed PCR with
primers specific for the rtTA on cDNA from bone samples
of bi-transgenic animals, tetop-LAG608G+;a1p-rtTA+
(Figure 1). We also included cDNA samples from a
different rtTA line (SM22-rtTA)  as a positive control
for the PCR assay (Figure 1). Whereas the positive
control sample had a fragment of expected size, there was
no amplification in any of the bone samples from
a1prtTA+ transgenic mice, indicating that the transactivator
was not expressed in bone (Figure 1).
Mouse genetic background and transgene integration
have been discussed as possible factors for variation in
expression levels of transactivator and target gene
transcription [23-25]. To test for differences in expression of
the transgenes in mice with different genetic
background, we performed PCR on cDNA from
tetopLAG608G+;a1p-rtTA+ bi-transgenic mice that were on
pure C57BL/6, pure FVB/NCrl or mixed C57BL/6;FVB/
NCrl background. While the positive control sample,
with cDNA from bone of bi-transgenic mice
tetopLAG608G+;Sp7-tTA+, gave a strong amplification of both
the human lamin A and laminAdel 150 transcripts,
there was again only very weak amplification in samples
from tetop-LAG608G+;a1p-rtTA+ bi-transgenic mice on
different genetic backgrounds (Figure 1).
Because the PCR assay that analyzed the expression of
the transactivator was not cDNA-specific  and could
amplify genomic DNA, we also included a RNA sample
and DNA samples of increasing concentrations from
bitransgenic tetop-LAG608G+;a1p-rtTA+ mice, to control
for genomic DNA contamination. Amplification was
seen for the cDNA sample of
tetop-LAG608G-;SM22rtTA+ and in genomic DNA samples that had ≥10 ng of
DNA template, but there was no amplification in the
RNA sample (Figure 1). This result indicated that there
was no transcriptional activity of the a1p-rtTA,
Figure 1 No detectable expression of transactivator and target gene. PCR on cDNA from bone (lanes 2-9 and lanes 11-13) and aorta (lane
10) was performed with primer pairs specific for the reverse transactivator. Respective genetic background is indicated for bi-transgenic
tetopLAG608G+;a1p-rtTA+ mice. No-reverse transcriptase cDNA sample (lane 11), no-template control (lane 12) and increasing concentrations of
genomic DNA template (1, 10, 20, and 50 ng in lanes 14-17, respectively) were included as controls for the PCR assay. PCR on a RNA sample
from bone (100 ng of total RNA was used as template for the PCR, lane 18) indicated that there was no significant genomic DNA contamination.
Lane 1 is a 100-bp ladder (Invitrogen). Presence and absence of transgene are indicated by + and -, respectively. LA, human lamin A, 276 base
pairs; LAdel150, progerin, 123 base pairs. RT-PCR for b-actin served as a control for the RT.
independent of genetic background. In addition, the
results also indicated that the positive control was an
accurate control, even though it amplified genomic
DNA, as there was no significant DNA contamination
in the RNA extracts and therefore the amplification
originated from cDNA.
To analyze the transgenic expression of human lamin
A and progerin at the protein level, we performed
western blot to screen for transgenic expression using an
antibody specific for human lamin A/C (and does not
cross-react with mouse lamin A/C) [10,26]. For
tetopLAG608G+;a1p-rtTA+, bone protein samples were
extracted for the different genetic backgrounds, pure
FVB/NCrl (n = 6) and C57BL/6J (n = 2) and mixed
FVB/NCrl;C57BL/6J (n = 15). For tetop-LAwt+;a1p-rtTA
+, protein samples were extracted from bones of mice
on the FVB/NCrl;C57BL/6J background (n = 7). Protein
extracts from bone of adult wild-type C57BL/6J,
tetopLAG608G+;Sp7-tTA+, tetop-LAG608G-;Sp7-tTA+ and
tetop-LAG608G-;Sp7-tTA- mice were included as
controls. No lamin A/C protein was detected in any
bitransgenic tetop-LAG608G+;a1p-rtTA+ or tetop-LAwt+;
a1p-rtTA+ or single-transgenic
tetop-LAG608G+;a1prtTA- or tetop-LAwt+;a1p-rtTA- mice, showing that the
very low levels of RNA transcription detected by
RTPCR were insufficient for protein translation (Figure 2
and data not shown).
In addition to western blot, we analyzed the transgenic
expression using immunofluorescence. Primary
osteoblasts were isolated from calvariae of 4-week-old
bitransgenic animals (tetop-LAG608G+;a1p-rtTA+, n = 14)
and single-transgenic control mice
(tetop-LAG608G+;a1prtTA-, n = 8) on both pure FVB/NCrl and pure C57BL/
6J backgrounds. Immunofluorescence staining of cells
prepared by cytospin with an antibody specific for
human lamin A/C did not detect protein expression in
osteoblasts isolated from tetop-LAG608G+;a1p-rtTA+
mice, regardless of genetic background (Figure 3a-d).
Osteoblasts isolated from tetop-LAG608G+;Sp7-tTA+
bitransgenic mice were included as controls, and
osteoblasts from these mice showed staining of the nuclear
lamina (Figure 3e-f).
Our expression analysis unequivocally showed that
there was no transgenic expression of human lamin A
Figure 2 The a1p-rtTA mouse failed to transactivate the lamin
A minigene. Western blot on bone protein extracts from
bitransgenic animals, tetop-LAG608G+;a1p-rtTA+ (lanes 2-4). Human
lamin A/C (mab3211) and b-actin (A5441) primary antibodies were
used. Protein extracts from a Hutchinson-Gilford progeria sample
AG03506 (lane 1) and bone protein extracts from tetop-LAG608G+;
Sp7-tTA+ mice (lane 6) and wild-type mice (lane 5) were included as
positive and negative controls, respectively. Presence and absence
of transgene are indicated by + and -, respectively
Figure 3 Lack of the human lamin A minigene protein product
in primary osteoblasts from tetop-LAG608G+;a1p-rtTA+
bitransgenic mice. No immunofluorescence staining was detected
when using an antibody specific for human lamin A/C on
osteoblasts isolated from bi-transgenic tetop-LAG608G+;a1p-rtTA+
mice on different genetic backgrounds, FVB/NCrl a and C57BL/6J c.
Positive staining was obtained using the same antibody on cells
from tetop-LAG608G+;Sp7-tTA+ bi-transgenic mice e. Merged panels
with DAPI b, d, f. Scale bars: 50 μm.
or progerin in bone or osteoblasts from bi-transgenic
mice, tetop-LAG608G+;a1p-rtTA+, and this is most likely
the result of a lack of expression of the transactivator.
Previous and on-going work in our laboratory, using
various target genes of human lamin A in combination
with a variety of inducible transcriptional activators for
directed expression to multiple tissues (skin, bone, and
smooth muscle), have been successful in creating
tissuespecific mouse models for HGPS [9, 10, 11 and
manuscripts in preparation]. Therefore, we postulate that the
reported malfunction of tetop-LAG608G+;a1p-rtTA+ as a
bone-specific mouse model is due to problems with
a1p-rtTA transgenic mice rather than with our human
lamin A minigenes. Reasons for the inadequate activity
of the transactivator presented in this study remain
unclear, and we did not perform any further
experiments to investigate the possible underlying mechanisms
that might have caused the inactivity of the promoter.
However, speculative reasons for defective
tetracyclineregulated activators have been discussed before [20,21].
Long-term instability because of epigenetic modification,
such as methylation [27,28], problems related to small
construct sizes, positional- and dosage-dependent effects
, and unexpected transgene expression pattern 
have been reported by various investigators [3,22]. The
tet-ON/OFF system is a powerful model system for
studying genes in vitro or in vivo. Previously constructed
transactivators are helpful tools within the scientific
community that can be easily shared to create new
model systems for use in different research contexts.
The purpose of this study is to report our negative
experience with the a1p-rtTA transgenic mice and to
reinforce the obligation of all investigators to monitor
the long-term stability of generated
tetracycline-controlled transactivators, both over the life-span of an
individual mouse and over multiple generations of founder
Acknowledgements and funding
This work was supported by grants from the Karolinska Institutet, the
Swedish Research Council, the Swedish Foundation for Strategic Research,
the Torsten and Ragnar Söderberg Foundation, the Tore Nilsson Foundation,
the Åke Wiberg Foundation, the Hagelen Foundation, the Loo and Hans
Osterman Foundation, the OE and Edla Johansson Foundation, and the Lars
Hiertas Minne Foundation. ES is supported by the Karolinska Institutet
Faculty Funds for doctoral students (KID).
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