Developmental Biology 325 (2009) 171–178

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Developmental Biology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / d e v e l o p m e n t a l b i o l o g y

rtTA toxicity limits the usefulness of the SP-C-rtTA transgenic mouse
Mitsuru Morimoto, Raphael Kopan ⁎Department of Developmental Biology and Department of Medicine, Division of Dermatology, Washington University School of Medicine, Saint Louis, MO 63110, USAa r t i c l e

i n f oArticle history:
Received for publication 3 July 2008
Revised 30 September 2008
Accepted 7 October 2008
Available online 1 November 2008
Keywords:
Doxycycline
Inducible conditional knockout
Genetic backgroundToxicity of transgene
Lung
AirwayOrganogenesis
Surfactant

a b s t r a c t
The doxycycline (DOX)-inducible gene expression systems allow tight temporal and spatial control oftransgene expression, invaluable in studies of organ development and disease pathogenesis. Transgenic miceusing the human Surfactant Protein C promoter to drive the expression of the reverse tetracyclinetransactivator (SP-C-rtTA) enabled functional analysis of essential gene function during lung development.
Here we report that DOX-fed SP-C-rtTA mice during the period in which Type II cells differentiate results incellular toxicity that may have confounded the interpretation of previous reports using this line. These effects
included impaired alveologenesis, loss/reduction in expression of surfactant-associated proteins, and death.
Severity was dependent on genetic background: outbred mice or those on a CD1 background are highly
susceptible, whereas the C57BL/6 background appeared resistant by morphological criteria. However,quantitative analysis reveled that DOX-fed, SP-C-rtTA C57BL/6 pups had reduced surfactant mRNA
accumulation that could contribute to synthetic lethality when combined with other genetic alterations.
We conclude that the combination of genetic backgrounds, length of DOX exposure and the presence of the
SP-C-rtTA transgene contributed more than previously appreciated to the similarities seen in the phenotypes
reported by investigators using the SP-C-rtTA, (tetO)7-Cre. These studies demonstrate the importance ofusing appropriate SP-C-rtTA only controls in all experiments.
© 2008 Elsevier Inc. All rights reserved.

Introduction
Conditional gene inactivation has evolved into a standard method
used to decipher the function of essential genes in any desired organ.
In principle, all methods rely on expression of Cre recombinase toremove “ﬂoxed” alleles, or genomic DNA fragments bracketed by loxPsites. The combination of cell type speciﬁc promoters, which provide
spatial speciﬁcity for Cre expression, with inducible promoters, which
endow the investigator with temporal control, contributed to theprevalence of this technology.
Cre toxicity is becoming appreciated by a growing number ofinvestigators. The ﬁrst reports came from the Berns (Akagi et al., 1997)
and Capecchi (Schmidt-Supprian and Rajewsky, 2007) labs, whichshowed that somatic recombination can occur in the absence of loxPsites. This was thought to be mitigated by selection of tolerated Crelevels in the process of generating Cre-transgenic mice. However,another “Toxic alert” has recently been raised (Editorial, 2007). Lee
et al. (Lee et al., 2006) have shown that RIP-Cre mice, used for
exploration of genetic pathways controlling pancreatic beta-celldevelopment and function, display a phenotype of glucose intolerance
even in the absence of any ﬂoxed allele. Results from studies of thesemice were reported in 21 publications prior to this discovery. Others
have also argued that toxicity can confound the results of experiments
not controlling for Cre alone (Schmidt-Supprian and Rajewsky, 2007).
⁎ Corresponding author.
E-mail address: Kopan@wustl.edu (R. Kopan).
0012-1606/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2008.10.013

Bi-transgenic systems, driving expression of rtTA from tissue
speciﬁc promoters and activating Cre with a doxycycline/rtTAinducible promoter ((tetO)7-Cre transgene), have emerged as a leading
system permitting tight temporal and spatial control of gene mutation(Branda and Dymecki, 2004). In the pulmonary research ﬁeld, reverse
tetracycline transactivator (rtTA) may be driven by the humanSurfactant Protein C (SP-C) promoter in SP-C-rtTA transgenic mice
(Perl et al., 2002a,b). Cre expression is activated after animals also
carrying (tetO)7-Cre are provided with doxycycline-containing food orwater (Perl et al., 2002b). At present, the standard method to achieve
gene loss in conduct airway and alveolar epithelial cells during desired
developmental stages is to administer doxycycline (DOX) to thepregnant tri-transgenic dam which, in addition to SP-C-rtTA and(tetO)7-Cre, carries a ﬂoxed allele (Sauer, 1998).
In preparing to investigate the role of Notch signaling (ArtavanisTsakonas et al., 1999; Ilagan and Kopan, 2007) in airway epithelial
cells, we administered doxycycline to tri-transgenic SP-C-rtTA,(tetO)7-Cre, RBPjkﬂox/ﬂox mice. To our surprise, we found that outbred
controls carrying SP-C-rtTA died at birth with impaired alveologenesis
and reduced surfactant production, independent of (tetO)7-Cre
inheritance. We discovered that strain-dependent sensitivity of lungType II epithelial cells to SP-C-rtTA, peaking between days 15.5 and18.5 of gestation, is responsible for this lethality. CD1 mice wereseverely affected whereas the inbred C57BL/6 strain was affected to alesser degree that might nonetheless synergize with a targeted allele.
The considerable toxicity of the rtTA/DOX combination during thedevelopment of distal lung epithelial cells led us to review the172

M. Morimoto, R. Kopan / Developmental Biology 325 (2009) 171–178

literature in which SP-C-rtTA, (tetO)7-Cre transgenic mice were used.
We noticed an alarming similarity in phenotypes that could be
attributed at least in part to the toxic effects of rtTA, warranting reexamination of this popular targeting method.
Materials and methods
Generation of SP-C-rtTA, (tetO)7-Cre, RBPjkﬂox/ﬂox mice and doxycycline
administration
RBPjkﬂox/ﬂox mice were kindly provided by Dr. T. Honjo, Kyoto
University (Tanigaki et al., 2002). SP-C-rtTA, (tetO)7-Cre mice weregenerated by Dr. J. Whitsett at the University of Cincinnati, provided to
us in mixed background of C57BL6/CD1. PCR analysis conﬁrmed that
this rtTA gene contains the C-terminal fragment of the VP 16
transcription activator (data not shown). RBPjkﬂox/ﬂox mice in a similar
background were mated with SP-C-rtTA, (tetO)7-Cre mice to produce
SP-C-rtTA, (tetO)7-Cre, RBPjk+/ﬂox mice that were then mated with
RBPjkﬂox/ﬂox mice to obtain SP-C-rtTA, (tetO)7-Cre, RBPjkﬂox/ﬂox males.
These were mated with RBPjkﬂox/ﬂox females to produce animals foranalysis. Pregnant females were fed doxycycline food (600 mg/kg)(Bio-Serv) from E8.5 to E18.5 or E15.5. The mating strategy isillustrated in Supplemental Table 1A. Littermates were compared toeach other in individual experiments. All animal procedures were
performed according to NIH guidelines and maintained in the animal
facility under Washington University animal care regulations.
Survival assay
We dissected doxycycline-fed mice at E18.5 to collect fetuses. The
umbilical cord was cut near the placenta with scissors to avoid excess
bleeding. Amniotic ﬂuid and membranes were removed from themouth and nose using a cotton swab. Body temperature was kept
elevated by maintaining the pups on a heat plate at 37 °C. Pups chests
were depressed with a cotton swab to stimulate breathing.
Histology and immunohistochemistry
The lungs were dissected and ﬁxed in 4% paraformaldehyde (PFA),
embedded in parafﬁn and sectioned at 6 μm. The sections were thenstained with Hematoxylin and Eosin (H&E) for histological analysis.
For immunohistochemistry, the sections were boiled in Trilogy (Cell
Marque) or Antigen Unmasking Solution (Vector Laboratories, Inc) for
antigen retrieval, pre-blocked with PBS supplemented with 1% BSA,
0.2% skimmed milk, and 0.3% Triton X-100. A rabbit primary anti-Prosurfactant Protein C (1:2000; Chemicon) or Aquaporin5 antibody(1:200; Chemicon) was used, followed by a biotinylated anti-rabbit
IgG (1:1000). Vectastain ABC kit (Vector Laboratory, Inc) and DAB
Substrate Kit (Vector Laboratory, Inc) were used to detect the signal.
RNA isolation and quantitative analysis
Fresh lung samples were immediately immersed in RNAlater(Ambion, TX) and homogenized by electric homogenizer at
12,000 rpm for 30 s. Total RNA was collected by using RNeasy kit(QIAGEN Sciences) according to the manufacturer's speciﬁcations. The
collected RNA was used for cDNA preparation using SuperScript II RT
with random hexamers (Invitrogen). The cDNA was ampliﬁed withSYBR-Green PCR Master Mix (Applied Biosystems, CA) and genespeciﬁc exon-exon junction-spanning primers using the ABI7700
sequence detection system (Applied Biosystems, CA). Normalizations
across samples were performed using glyceraldehyde-3-phosphate
dehydrogenase (Gapdh) primers. The differences between transgenic
samples and RBPﬂox/ﬂox or Wild Type as controls were calculatedusing the comparative CT(2−ΔΔCT) method. The histograms in theﬁgures show the mean ± s.e.m. of one to three independent experi-ments, each of which was performed in triplicate. All qRT-PCR primers
were tested for the presence of non-speciﬁc primer dimers and forlinear ampliﬁcation. Primer sequences are below,
Sftpa; AGGCAGACATCCACACAGCTT and ACCAGTGGTTTCTCCCAATCAC, Sftpb; GGAACACCAGTGAACAGGCTATG and AAACTGTTCACACTTTTGCCTGTCTA, Sftpc; ACCCTGTGTGGAGAGCTACCA andTTTGCGGAGGGTCTTTCCT, Sftpd; CCAACAAGGAAGCAATCTGACAT and
CAAGACAAGCATGGAGAGAAAGG, ABCa3; AAGCCTGAACGAGTATGGTAGA and CTCCTCTAGGTCACCTAGCAC, Cebpa; ACAAGAACAGCAACGAGTAC C a n d GT C A A C T C C AG C AC C T T C T G , a n d GA P D H ;AATGTGTCCGTCGTGGATCTGA and GATGCCTGCTTCACCACCTTCT.
Results and discussion
SP-C-rtTA/DOX combination leads lethal defectIn order to generate conditional knockout mice in which the RBPjk
gene, a core component of Notch signaling, is deleted from the lungepithelial cells, RBPjkﬂox/ﬂox females were mated to SP-C-rtTA, (tetO)7Cre, RBPjk+/ﬂox males. The pregnant dams were maintained on DOX(600 mg/kg) diet from 8.5 days post coitum (dpc), a day before lungdevelopment starts in the developing embryos. To examine deletion
efﬁciency in utero, and to record the consequences of RBP loss, litterswere delivered by cesarean section at E18.5. We noticed that some
pups died within 1 h, displaying respiratory distress (Supplemental
Table 1A). These pups failed in inﬂating their lungs while theirlittermates breathed normally (Supplemental Movie 1). Surprisingly,
double-transgenic SP-C-rtTA, RBPjkﬂox/ﬂox pups were found among thedead; in fact, all dead animals in this and ﬁve additional littersinherited SP-C-rtTA but only some inherited Cre or RBPjk alleles(Supplemental Table 1B). The observation that Cre inheritance was nota factor in lethality, but rtTA inheritance was, is a strong indicator of
toxicity associated with the SP-C-rtTA transgene when the dams areprovided with 600 mg/kg DOX in their diet.
Whereas RBPjk heterozygous mice generally do not display aphenotype, histological analysis revealed enlargement of the alveolar
space in embryos inheriting the SP-C-rtTA transgene and exposed toDox (Figs. 1A–D). Furthermore, the distributions of Type I and Type II
cells in the alveolar epithelium was impaired as determined byimmunohistochemistry with Aquaporin 5 (Figs. 1E–H) and ProSP-C
antibodies (Figs. 1I–L). Since infant mortality is often associated with
disruption of Surfactant-associated proteins (Sftp) secretion fromType II cells, we estimated the expression levels of Sftp A, B, C and D(Whitsett et al., 2005), their transporter ABCa3 (Ban et al., 2007;
Cheong et al., 2007; Fitzgerald et al., 2007) and transcriptional
regulator C/EBPα (Flodby et al., 1996; Sugahara et al., 2001) using qRTPCR (Fig. 1M). In comparison with RBPjk+/ﬂox, expression of thesefactors was reduced signiﬁcantly in all Dox-fed embryos inheriting the
SP-C-rtTA. This quantitative analysis conﬁrmed that Cre inheritance
did not contribute to the effect. These data therefore indicate that thepresence of the SP-C-rtTA transgene is the genetic determinantcausing abnormal alveolar development in embryos exposed to DOX,
and that Type II cells are the main cell types affected. Since Type II cells
are progenitors for Type I cells (Adamson and Bowden, 1974), theabnormal alveoli are likely the result of the Type II cell loss.
Lethality is due to tTA/DOX toxicity at late developmental stage
Type II cells appear in distal airways from E16.5, known as thecanalicular stage, and produce surfactant proteins as they mature.
Since exposing embryos to DOX from E8.5 to E15.5 may be sufﬁcient toachieve complete deletion of ﬂoxed alleles, we wanted to ask if
removing DOX during the period in which Type II cells differentiate
would eliminate the toxicity associated with the rtTA/DOX complex.
When we administered DOX from E8.5 to E15.5, we rescued doubleand triple-transgenic inheriting SP-C-rtTA (Supplemental Table 1B).

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173

Fig. 1. Doxycycline administration to the triple-transgenic mice leads to impaired alveologenesis whether or not the (tetO)7-Cre transgene was inherited. (A–D) Lung sections
prepared at E18.5 and stained with the Hematoxylin and Eosin staining revealed alveolar enlargement in mice inheriting SP-C-rtTA (C and D) but not RBPjk+/ﬂox or (tetO)7-Cre, RBPjk+/ﬂox(A and B). Scale bar: 0.1 μm. Immunohistochemistry with anti-Aquaporin 5 (E–H) or anti-ProSP-C (I–L) antibody revealed defective differentiation of Type I cells (G and H) and Type II
cells (K and L) in mice inheriting rtTA. Scale bar: 0.05 μm. (M) Quantitative RT-PCR analysis revealed expression levels of Surfactant-associated protein panels A to D (Sftpa to d) andtheir regulators ABCa3, C/EBPα in each genotype. Each value was normalized to the internal control GADPH and standardized to RBPjk+/ﬂox. ⁎P b 0.05 versus RBPjk+/ﬂox.

The pups born with this treatment grew to adulthood without any
obvious respiratory problem. Histological analysis conﬁrmed that Cre
activity during the E8.5–E15.5 window was sufﬁcient to remove thetargeted alleles (Supplemental Fig. 1A).
At the histological level, double-transgenic mice treated from E8.5
to E15.5 exhibited lung morphology indistinguishable from wild typemice at E18.5 (Figs. 2A–D). However, although the distribution of TypeI and Type II cells was restored to normal (Figs. 2E–L), quantitative RTPCR analysis revealed suppressed expression of Sftps and theirregulators (Fig. 2M). Because SP-C-rtTA, RBPjkﬂox/ﬂox double-transgenic
mice displayed the same phenotype as their triple-transgenic, RBPjkdeﬁcient littermates (Supplemental Fig. 1A), we conclude that neither
Cre activity nor loss of Notch signaling in the distal airway contributes tothe observed phenotype (the full description of the phenotype will be
published elsewhere). These results reveled that although severe
phenotypes are caused by exposure to rtTA/DOX between E15.5 to
18.5 (the canalicular and saccular stages), a moderate phenotype
characterized by suppression of the Sftps and their regulators still

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M. Morimoto, R. Kopan / Developmental Biology 325 (2009) 171–178

Fig. 2. Rescue attained by suspension of doxycycline administration at E15.5. (A–D) Lung sections prepared at E18.5 and stained with the Hematoxylin and Eosin staining weremorphologically normal. Scale bar: 0.1 μm. Normal distribution of Type I cells and Type II cells were observed with immunohistochemistry for Aquaporin 5 (E–H) and ProSP-C (I–L).
Scale bar: 0.05 μm. (M) Expressions of Sftpa, b, d and ABCa3 were still reduced while Sftpc and Cebpa were partially restored in the rescued the SP-C-rtTA mice. Each value wasnormalized to the internal control GADPH and standardized to RBPjkf/f. ⁎P b 0.05 versus RBPjkf/f.

occurs in the transgenic mice inheriting the SP-C-rtTA transgene. Theseobservations confound the interpretation of studies using this line due
to possible synergy between the toxic effects of SP-C-rtTA on Type II cells
and the induced gene ablation.
The particular genetic background appears resistant to the rtTA/DOX
toxicity
Genetic background can be another confounding factor inﬂuencing the phenotype of a transgenic mouse. Like many studies

involving multiple transgenes, we analyzed the behavior of SP-CrtTA on a mixed background, here containing genetic contribution
from C57BL/6 and CD1 with minimal contribution from FVB (thestrain in which SP-C-rtTA and (tetO)7-Cre were made) and 129Sv(used to generate RBPjk+/ﬂox). To ask if the genetic backgroundcontributed to rtTA/DOX toxicity, we mated an SP-C-rtTA, (tetO)7Cre, RBPjk+/ﬂox sire with wild type females of C57BL/6 or CD1
background. We fed the pregnant dams doxycycline diet from E8.5
to E18.5, delivered their fetuses by cesarean section at E18.5 andexamined the phenotypes. Interestingly, alveolar morphology wasM. Morimoto, R. Kopan / Developmental Biology 325 (2009) 171–178

normal in all SP-C-rtTA pups on the C57BL/6 background (Figs. 3C, D)
whereas it was clearly abnormal on the CD1 background (Figs. 3G,
H). Likewise, Type I and Type II cells were lost on the CD1background but their distribution was normal on the C57BL/6
background (Figs. 3K, L, O, P, S, T, W and X). CD1 pups inheriting the175

SP-C-rtTA transgene exhibited reductions in the level of surfactants
and their regulators comparable to that in mixed backgroundanimals (compare Figs. 1M, 2M and 4B). Although lung morphology
with increased C57BL/6 background appeared similar to nontransgenic wild type (Fig. 4A), a signiﬁcant reduction in theFig. 3. The sensitivity to the toxic effects of rtTA depends on genetic background. Lung sections were prepared from offspring of C57BL/6 (A–D, I–L and Q–T) or CD1 (E–H, M–P and U–X) dams. (A–H) The Hematoxylin and Eosin staining demonstrated alveolar enlargement in SP-C-rtTA transgenic mice on the CD1 genetic background (G and H) but not the C57BL/6
background (C and D). The impaired distribution of Type I cells (I–P) and Type II cells (Q–X) was observed by immunohistochemistry in only the CD1 genetic background (O, P, W andX) but not the C57BL/6 background (K, L, S and T).

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M. Morimoto, R. Kopan / Developmental Biology 325 (2009) 171–178

Fig. 4. Genetic background inﬂuences surfactant production in SP-C-rtTA transgenic mice with doxycycline administration. (A) Mating with a wild type C57BL/6 female reduced the
toxicity SP-C-rtTA transgenics from the severe suppression of the expression of the surfactants and surfactant-related factors. However, the expression of Sftpa, d and ABCa3 were still
relatively repressed. (B) In the CD1 background, the SP-C-rtTA transgenic group showed suppression of gene expression similar to the previous experiments in Figs. 1M and 2M. Eachvalue was normalized to the internal control GADPH and standardized to wild type. The SP-C-rtTA transgene is sufﬁcient to reduce their expression. ⁎P b 0.05 versus wild type.

expression of SftpA, D and ABCa3 was still detected. These data
indicate that the outbred CD1 background and the CD1; C57BL/6
mixed animals were susceptible to rtTA/DOX toxicity. It is important
to note that even on the C57BL/6 background, the potential for
confounding impact from rtTA/DOX toxicity exists.
rtTA/DOX toxicity may have confounded the interpretation of previous
reports
Identifying the precise mechanism behind SP-C-rtTA toxicity, or
the genes conferring resistance, is outside the scope of this report andof questionable relevance to developmental biology or human health.
However, several mechanistic assumptions can be made. The rtTAVP16 used in these strains is known to bind to pseudo-tetO promoter
sequence and may even bind non-speciﬁcally in a concentration and
DOX dependent manner (Urlinger et al., 2000). Since global rtTA

expression under CMV promoter does not impair mouse developmentin the presence of DOX (Furth et al., 1994), one can assume that
although the strength of the SP-C promoter coupled with transgene
copy number produces tolerable amounts of this repressor, addition of
DOX during the developmental window deﬁned in our study
represses genes essential for Type II cell development, resulting intoxicity and death.
It should be noted that others reported toxicity of rtTA to Clara cells
in adult lungs when using the CCSP-rtTA transgene even in theabsence of DOX (Sisson et al., 2006), leading to an emphysema-likephenotype and a caution regarding potential toxicity (Whitsett andPerl, 2006). If SP-C-rtTA were similarly toxic to mature Type II cells,
dams would suffer respiratory distress; they did not. This would beconsistent with toxicity following periods of maximal rtTA expression.
Given the widespread use of these rtTA lines, it would be prudent toassume that other embryonic and adult cell types are sensitive to rtTA

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177

Table 1
Type II cell phenotypes and survival in published studies that have used the SP-C-rtTA, (tetO)7-Cre transgenic mice for gene targeting
Targeted gene

Alveolar morphology

Surfactant reduction

AQP5 defectSurvival at P1

DOX administration

Genetic backgroundRef.

b-catenin
Foxa2
Foxa1−/−, Foxa2
Laminin a5
Cebpa
Cebpa
STAT3
Calcineurin b1

Severe enlargement
Atelectasis
AbnormalEnlargement
Atelectasis
Atelectasis
Atelectasis
Enlargement

Yes
Yes
Yes
Yes
SftpC No SftpA, B Yes
Yes
No
Yes

N.A.
Yes
Yes
Yes
Yes
Yes
N.A.
Yes

625 mg/kg diet, E0.5∼
25 mg/g diet, E0.5∼625 mg/kg diet, E0.5∼
1 mg/ml water, E6.5∼
625 mg/kg diet, E0.5∼
1 mg/ml water, E0.5∼625 mg/kg diet, E0.5∼
N.A.

C57BL/6; CD1
N.A.
N.A.
C57BL/6; CD1
N.A.
C57BL/6; FVB/N
N.A.
C57BL/6; FVB/N

Mucenski et al., 2003
Wan et al., 2005
Wan et al., 2004
Nguyen et al., 2005
Basseres et al., 2006
Martis et al., 2006
Hokuto et al., 2004
Dave et al., 2006

PTEN
RBPjk

Atelectasis
Enlargement

Yes
Yes

Yes
Yes

None
46% Survival
N.A.
None
14% Survival
None
100% Survival
100% Survival(but all dead at P2)
10% Survival
33% Survival

1 mg/ml water, E10.5–16.5
625 mg/kg diet, E8.5∼

C57BL/6; 129Ola
C57BL/6; CD1

Yanagi et al., 2007
Table S1

NA., information not available.

alone or in combination with DOX, requiring more detailed molecular
control experiments than previously deemed necessary (Whitsett andPerl, 2006).
A case in point are studies using the SP-C-rtTA, (tetO)7-Cre system
employed to date in at least nine publications (Basseres et al., 2006;
Dave et al., 2006; Hokuto et al., 2004; Martis et al., 2006; Mucenski etal., 2003; Nguyen et al., 2005; Wan et al., 2005, 2004; Yanagi et al.,2007) (Table 1). These studies have yielded important new insights
into genetic pathways controlling the development, function andmaintenance of lung epithelial cells. However, in addition tophenotypes speciﬁc to the targeted allele, all studies reported similar
reductions of surfactant-associated protein and Aquaporin-5 (withthe exception of STAT3ﬂox/ﬂox (Hokuto et al., 2004)).. We establishedthat loss of Type II cells and their products is the hallmark of rtTA/
DOX poisoning. The authors of the STAT3 targeting study have notreported the genetic background of their animals but we expect thatthese mice were on a genetic background resistant to toxicity.
Likewise, it is tempting to point out that the lethality reported inTable 1 might depend on genetic background and not on geneablation since all mice with a CD1 contribution display some degreeof perinatal mortality. Moreover, rtTA/DOX toxicity and backgroundcontribution could help explain how two independent studies
targeting C/EBPα reported slightly different outcomes (Basseres etal., 2006; Martis et al., 2006).
Conclusions
While interpretation of past reports pertaining to Type II cell
development is confounded by the rtTA/DOX toxicity, additional
controls should be able to identify the phenotypes obtained with SPC-rtTA mice that are dependent on rtTA toxicity, gene ablation orinteraction between the two. It is important to note that even when
SP-C-rtTA littermates are morphologically indistinguishable from wild
type, our data indicates that SP-C-rtTA transgenic mice are compromised— they have reduced mRNA levels for surfactants, theirregulators, and an unknown number of other genes (Figs. 1, 2 and
4). The rtTA system in general, and the SP-C-rtTA transgene inparticular, are powerful tools that will be difﬁcult to replace. Until aperfect system is devised, proper controls must include paralleledmolecular analysis of DOX-fed rtTA strains in matched background totheir bi- and tri-transgenic littermates.
Acknowledgments
The authors thank Drs. Adrian Shifren, David Ornit, and Jeffery
Miner for careful reading of the manuscript. We also thank Dr. Jeffery
Whitsett for SP-C-rtTA, (tetO)7-Cre mice. This work was supported by
Washington University. MM was supported by a Toyobo Biotechnology Foundation Long-term Research Grant and the Japanese Society
for the Promotion of Science. RK was supported by NIH grant HD
044056.

Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ydbio.2008.10.013.

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