Conditional gene expression in the mouse inner ear using Cre-loxP.

Abstract

In recent years, there has been significant progress in the use of Cre-loxP technology for conditional gene expression in the inner ear. Here, we introduce the basic concepts of this powerful technology, emphasizing the differences between Cre and CreER. We describe the creation and Cre expression pattern of each Cre and CreER mouse line that has been reported to have expression in auditory and vestibular organs. We compare the Cre expression patterns between Atoh1-CreER(TM) and Atoh1-CreER(T2) and report a new line, Fgfr3-iCreER(T2), which displays inducible Cre activity in cochlear supporting cells. We also explain how results can vary when transgenic vs. knock-in Cre/CreER alleles are used to alter gene expression. We discuss practical issues that arise when using the Cre-loxP system, such as the use of proper controls, Cre efficiency, reporter expression efficiency, and Cre leakiness. Finally, we introduce other methods for conditional gene expression, including Flp recombinase and the tetracycline-inducible system, which can be combined with Cre-loxP mouse models to investigate conditional expression of more than one gene.

Full text

Page 1

Invited Review
Conditional Gene Expression in the Mouse Inner Ear
Using Cre-loxP
BRANDON C. COX1, ZHIYONG LIU1, MARCIA M. MELLADO LAGARDE1, AND JIAN ZUO1
1Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN
38105, USA
Received: 5 December 2011; Accepted: 19 March 2012
ABSTRACT
In recent years, there has been significant progress in
the use of Cre-loxP technology for conditional gene
expression in the inner ear. Here, we introduce the
basic concepts of this powerful technology, emphasiz-
ing the differences between Cre and CreER. We
describe the creation and Cre expression pattern of
each Cre and CreER mouse line that has been
reported to have expression in auditory and vestibular
organs. We compare the Cre expression patterns
between Atoh1-CreERTMand Atoh1-CreERT2and
report a new line, Fgfr3-iCreERT2, which displays
inducible Cre activity in cochlear supporting cells.
We also explain how results can vary when transgenic
vs. knock-in Cre/CreER alleles are used to alter gene
expression. We discuss practical issues that arise when
using the Cre-loxP system, such as the use of proper
controls, Cre efficiency, reporter expression efficiency,
and Cre leakiness. Finally, we introduce other meth-
ods for conditional gene expression, including Flp
recombinase and the tetracycline-inducible system,
which can be combined with Cre-loxP mouse models
to investigate conditional expression of more than
one gene.
Keywords: cochlea, conditional gene deletion
CreER, Cre efficiency, Cre recombinase, ectopic
gene expression, Flp recombinase, knock-in, LoxP,
reporter lines, Tet-on, Tet-off, transgenic, utricle,
vestibular
Abbreviations: ABR – Auditory brainstem response;
BAC – Bacterial artificial chromosome; βgal – Beta-
galactosidase; BMP – Bone morphogenetic protein;
BDNF – Brain-derived neurotrophic factor; CFP –
Cyan fluorescent protein; Col1A1–Alpha (1) collagen
promoter; Col2A1–Type II collagen promoter; DTA–
Diphtheria toxin fragment A; E – Embryonic day;
eGFP – Enhanced green fluorescent protein; ES –
Embryonic stem cells; GER–Greater epithelial ridge;
GFAP – Glial fibrillary acidic protein; GFP – Green
fluorescent protein; GOI–Gene of interest; Hsp90–
Heat shock protein 90; iCsp3 – Drug-inducible
dimerizable caspase 3; IRES – Internal ribosome
entry site; KOMP – NIH knockout mouse project;
LER – Lesser epithelial ridge; myo7a – Myosin VIIa;
NICD – Notch intracellular domain; P – Postnatal
day; PAC – Phage artificial chromosome; Pkd1 –
Polycystic kidney disease 1; Rb – Retinoblastoma
protein; RFP – Red fluorescent protein; rtTA –
Reverse tetracycline transactivator; SHH – Sonic
hedgehog; TRE – Tetracycline responsive element;
tTA – Tetracycline transactivator
INTRODUCTION
The use of knockout or germline deletion mice has
been extremely useful in the past few decades to
investigate the role of specific genes in tissues or organs,
including the inner ear; however, this approach deletes
the gene of interest (GOI) from every cell in the body
throughout the life of the mouse and ~15–20 % of
germline deletions result in embryonic lethality
(Zambrowicz et al. 2003). In addition, germline
deletion models identify only the earliest functions of
Correspondence to: Jian Zuo & Department of Developmental Neuro-
biology&St. Jude Children’s Research Hospital&262 Danny Thomas
Place, Memphis, TN 38105, USA. Telephone: +1-901-5953891; fax:
+1-901-5952270; email: jian.zuo@stjude.org
JARO (2012)
DOI: 10.1007/s10162-012-0324-5
D 2012 Association for Research in Otolaryngology
JARO
Journal of the Association for Research in Otolaryngology

Page 2

the GOI, with postnatal gene functions often masked by
embryonic effects. Germline gene inactivation can also
cause pleiotropic effects, where the deletion of a single
gene influences multiple phenotypes, again preventing
the determination of an individual GOI’s function in a
particular organ. Many genes play different roles in
various cell types as well as different roles during
embryonic development compared to postnatal
ages; thus, cell type specificity and temporal
control of gene expression (known as conditional
gene expression) is needed to fully understand
their function. Mouse models that allow condition-
al gene expression permit the discovery and
dissection of GOI functions in a manner that is
specific to a chosen cell type and throughout the
life of the mouse.
The most common method to alter gene expres-
sion in a conditional manner is the use of the Cre-loxP
system (Kwan 2002). Here, we discuss the basics of
Cre-loxP technology including the differences be-
tween Cre and CreER and the generation of knock-
in, conventional transgenic, and bacterial artificial
chromosome (BAC) transgenic lines. We list each
Cre/CreER mouse strain that has been published in
the inner ear, organized by when Cre expression
occurs (separating embryonic and postnatal ages) and
where Cre expression occurs (separating auditory,
vestibular, ganglionic, and non-sensory regions). We
describe in detail how each mouse line was created, its
Cre expression pattern, and the relevant biological
discoveries that have been made using each condi-
tional allele. The last section of the review discusses
practical issues that arise when using the Cre-loxP
system,suchastheuseofpropercontrols,Creefficiency,
reporter expression efficiency, and Cre leakiness. We
also discuss applications for fate mapping and mosaic
Cre expression patterns. Finally, we present the basics of
two other methods to control gene expression in a
conditional manner—Flp recombinase and the tetracy-
cline-inducible system. This review serves as a thorough
introduction to conditional gene deletion and its use in
inner ear research as well as a compilation of current
information for researchers who routinely use condi-
tional mouse models.
THE CRE-LOXP SYSTEM
Cre is an enzyme originally from P1 bacteriophage
that acts as a site-specific recombinase, recognizing a
short sequence of DNA called a loxP site. A loxP site is
a consensus 34-bp DNA sequence that is not present
in the mouse genome and has directionality. Cre-
mediated recombination of genes flanked by loxP sites
(also called a floxed sequence) can result in the
excision, inversion, or translocation of DNA depend-
ing upon the location and orientation of the loxP sites.
The most common use of Cre recombinase is to
excise or delete the floxed DNA sequence which
occurs when two loxP sites are on the same strand of
DNA and are in the same orientation (Nagy 2000).
Numerous studies have shown that Cre-mediated
recombination can occur in a variety of cell types
(Sauer et al. 1989; Kuhn et al. 1995; Kellendonk et al.
1999; Feltri et al. 1999; Nagy et al. 2000) and that only
a few Cre molecules per cell are needed to excise the
floxed DNA (Nagy 2000). It is important to note that
Cre-mediated recombination is a permanent deletion
of the floxed DNA, and if cell division occurs after
Cre-mediated recombination, all daughter cells will
inherit this gene deletion.
To allow for cell type-specific control of gene
deletions, mouse models have been created where
Cre expression is controlled by a cell type-specific
promoter. These lines can then be crossed with mouse
lines containing a relevant part of a GOI that is
surrounded by loxP sites in the genome to generate
conditionalgenedeletion(Fig.1A).Theoverexpression
or ectopic expression of a gene can also be induced
using the Cre-loxP system. In this case, a construct
containing a promoter and a floxed “stop” sequence
upstream of a GOI (i.e., promoter-loxP-stop-loxP-GOI) is
inserted into the genome; thus, only Cre+cells are able
to remove the “stop” sequence and overexpress or
ectopically express the GOI (Fig. 1B; Zuo 2002; Gao et
al. 2004). This strategy is also used for reporter alleles so
that lacZ, green fluorescent protein (GFP), or other
fluorescent molecules are expressed in Cre+cells only
when the floxed “stop” sequence is removed.
To gain temporal control of gene expression, the
ligand-binding domain of an engineered steroid
hormone receptor is fused to the Cre enzyme and
the Cre molecules are sequestered in the cytoplasm by
heat shock protein 90 (Hsp90), keeping Cre inactive.
The translocation of the Cre–hormone receptor
fusion protein to the nucleus, which allows Cre to
become active, only occurs in a ligand-dependent (or
inducible) manner (Fig. 1C; Feil et al. 1996; Hayashi
and McMahon 2002). To prevent Cre activation by
endogenous steroid hormones, the engineered ste-
roid hormone receptor contains specific point muta-
tions that make it insensitive to endogenous steroid
hormones while retaining binding affinity for synthet-
ic analogues. The most common use of this strategy is
CreER alleles, where an altered ligand-binding do-
main of the estrogen receptor is fused to Cre and
mediates tamoxifen-dependent translocation to the
nucleus, but is insensitive to endogenous estradiol
(Hayashi and McMahon 2002). Therefore, only
exposure to tamoxifen will induce Cre activity allow-
ing temporal control of gene expression. Note that
tamoxifen is a prodrug that is hydrolyzed by the liver
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Page 3

to its active form, 4-hydroxy-tamoxifen (Furr and
Jordan 1984); thus, in vitro induction of CreER
requires the use of 4-hydroxy-tamoxifen instead of
tamoxifen to activate CreER. Two mutations in the
estrogen receptor are commonly used to generate
CreER alleles. CreERTM(also called CreERT) contains
a single-point mutation, G521R, in the ligand-binding
domain of the estrogen receptor, whereas CreERT2
contains a triple mutation (G400V, M543A, and L544A)
in this region (Feil et al. 1997; Indra et al. 1999). The
triple mutation results in a four to tenfold increase in
the sensitivity of CreERT2to 4-hydroxy-tamoxifen com-
pared to CreERTM; thus, in theory, less tamoxifen is
needed to induce Cre activity for CreERT2alleles
FIG. 1.
CreER. A Diagram of Cre-mediated deletion of a GOI flanked by
loxP sites. B Diagram of a reporter allele where Cre-mediated
deletion of a floxed “stop” sequence results in the expression of
Cre-mediated excision and the mechanism of inducible lacZ. C Diagram of inducible CreER-mediated excision. In the
absence of tamoxifen, CreER is sequestered in the cytoplasm by
Hsp90. In the presence of tamoxifen, CreER is translocated to the
nucleus where it recognizes loxP sequences and cleaves the DNA.
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Page 4

(Danielian etal. 1998; Indraetal.1999;Lietal.2000).It
is important to remember that many other factors may
affect Cre activity, such as the activity of the promoter
driving Cre expression at the time of induction, so the
doseoftamoxifenneededtogetoptimalresultsforeach
CreER line needs to be empirically tested with a
reporterline.OtherinducibleCrealleles,suchasCrePR
where the ligand-binding domain of the progesterone
receptor is fused to Cre, also exist, but are used less
frequently (Wunderlich et al. 2001).
GENERATION OF Cre/CreER LINES
Cre/CreER lines are generated by inserting the Cre
coding sequence into the endogenous coding region of
a gene to “highjack” the promoter of interest (i.e.,
knock-in) or by creating a transgene by randomly
inserting into the genome a fragment of DNA that
contains the promoter of interest (and/or enhancer
regions) followed by the Cre coding sequence. Conven-
tional transgenic lines use minimal promoter regions
(G10 kb), while BAC transgenic lines contain large
fragments of DNA (9100 kb). There are pros and cons
for each of these methods that affect the expression
pattern and expression level of Cre/CreER.
Knock-in alleles use homologous recombination in
embryonic stem (ES) cells to insert the Cre coding
sequence into the endogenous locus of the GOI. This
techniqueisexpensiveand time-consuming ashundreds
ofEScellsneedtobescreenedtofindthehandfulwhere
homologous recombination occurred. However, the
knock-in approach is advantageous because it is more
likely that the endogenous gene expression pattern will
be reproduced by theCre expression pattern (Tian et al.
2006). If the Cre coding sequence replaces or disrupts
the endogenousgene sequence,knock-inalleles arenull
alleles; thus, only heterozygotes can be used for condi-
tionalgeneexpression.Inthiscase,itisalsoimportantto
consider that a gene may be haploinsufficient, generat-
ing a phenotype when only one allele is present and
complicating the conclusions made from the deletion of
a GOI. When using such knock-in alleles, it is best to
compare results to controls that use the same Cre allele
without the floxed GOI to test for potential haploinsuffi-
ciency phenotypes.
To prevent a knock-in allele from generating a null
allele, an internal ribosome entry site (IRES) can be
used to drive Cre expression. The translation of mRNA
is generally dependent upon 5′ cap-mediated transla-
tion; however, the addition of IRES promotes an
internal initiation of translation, allowing a second
protein, such as Cre, to be made (Mountford and Smith
1995; Martinez-Salas 1999). Inmost cases,the IRES-Cre
constructisaddedtothe3′-endoftheGOIafterthestop
codon.ItisimportanttonotethattheefficiencyofIRES-
mediated translation varies according to cell type and
strain background (Kazadi et al. 2008).
The generation of transgenic lines is much faster and
cheaper than knock-in alleles, but is dependent on
having a well-defined promoter region. Even then,
other regulatory elements outside the promoter region
may be needed to recapitulate the endogenous gene
expression pattern; thus, many transgenic lines have
unexpected Cre expression patterns. In addition, trans-
genes are randomly inserted into the genome, which
can cause variation in Cre expression due to the effects
of enhancers or repressors in the local genomic region
(calledpositionaleffects).Thus,multiplefoundersneed
to be analyzed for each transgenic line created (Zuo
2002; Tian et al. 2006). The addition of insulators (i.e.,
an intron region of the chicken beta-globin gene) in
front of the transgenic promoter may lessen these
positional effects (Burgess-Beusse et al. 2002). Some-
times the insertion of a transgene into the genome can
inactivate an endogenous gene, so it is best to use
transgenic Cre lines as heterozygotes. Since convention-
al transgenes are relatively small, multiple copies of the
transgene are commonly inserted into the genome at
the same site or at multiple insertion sites. This can
increase the level of Cre expression and thus affect the
pattern of Cre expression. In addition, multiple trans-
gene copies can be reduced after successive generations
and cause genetic drift of Cre expression.
In contrast, BAC transgenic lines contain very large
segments of DNA that should include all regulatory
elements necessary to recapitulate the endogenous
gene expression pattern driven by the promoter of
interest. Although BAC transgenes are not subject to
strong positional effects, which often occur in conven-
tional transgenic lines, they are well known to rearrange
orbreakapartwhenintegratedintothehostgenome.In
fact, rearrangements can even occur in offspring after
germline transmission is obtained; thus, Cre expression
patterns may be different between founder lines and
successive generations. BAC transgene insertion into
the genome can also inactivate endogenous genes, so it
is also wise to use these alleles as heterozygotes. It is also
important to note that BAC vectors may contain
additional loxP sites. If just one loxP sequence is retained
in the BAC cloning vector, it can be transferred into the
mouse genome and can affect the outcome of Cre-
mediated excision. In addition, the large size of BAC
transgenes increases the likelihood that other genes
and/or promoter elements are within these transgenes
(Tian et al. 2006; Yu and Zuo 2009).
REPORTER LINES
To determine the pattern of Cre activity and to
quantify the percentage of Cre+cells, reporter mouse
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Page 5

lines are used. The most commonly used line is the
ROSA26LacZreporter (also called ROSA26R or R26R),
where lacZ, encoding β-galactosidase (βgal), is
expressed in Cre+cells after the removal of the floxed
“stop” sequence (Soriano 1999). βgal expression can
only be visualized in the presence of its substrate, X-
gal, or using βgal antibodies. Many other reporter
lines exist that use the ROSA26 locus or the CAG
promoter (a combination of the CMV enhancer with
the chicken β-actin promoter) such as ROSA26eGFP
(Giel-Moloney et al. 2007), ROSA26eYFP(Srinivas et al.
2001), CAGlacZ(Akagi et al. 1997), and CAGeGFP
(Kawamoto et al. 2000). In theory, a reporter line
driven by the ROSA26 locus or a CAG promoter is
ubiquitously expressed, and recent work has demon-
strated that the CAG promoter is eight to tenfold
stronger than the ROSA26 promoter (Nyabi et al.
2009; Chen et al. 2011).
xA more complex reporter line that is commonly
used is Z/EG. Here, the CAG promoter is used to
express lacZ in all cells until Cre-mediated recombi-
nation deletes the floxed lacZ gene, allowing the
expression of a second reporter, enhanced GFP
(eGFP) (Novak et al. 2000). Since eGFP has endoge-
nous fluorescence, Cre activity can be monitored in
live samples and Cre+live cells can be isolated using
FACS. Other reporter lines that use the same strategy
are the Z/AP line, where human alkaline phosphatase
is expressed after the deletion of the floxed lacZ
sequence (Lobe et al. 1999), and the mT/mG line,
where membrane-targeted GFP is expressed after the
deletion of a floxed membrane-targeted tdTomato
(Muzumdar et al. 2007).
Imaging the fluorescent reporter molecule does
not reflect the current expression of Cre, but instead
provides a history of Cre expression since the progeny
of Cre+cells will permanently express the reporter
regardless of whether they currently express Cre. In
addition, it is important to consider that it takes time
for the Cre enzyme to be translated and translocated
into the nucleus for recombination to occur (Nagy
2000). It takes additional time for the reporter protein
to be synthesized at a level high enough for detection.
Several groups have estimated this to take approxi-
mately 12 h by comparing the expression of the cell
type-specific promoter driving Cre with reporter
expression (Bouchard et al. 2004; Matei et al. 2005).
The delay for inducible CreER alleles occurs for
another reason. After injection, it takes time for
tamoxifen to enter the blood stream, be hydrolyzed
to its active form, reach cells in the inner ear, and
activate the CreER molecule for translocation to the
nucleus. Cre-mediated recombination has been
detected at the level of the genome 24–30 h after
tamoxifen injection (Weber et al. 2008); however,
detection using different reporter lines may vary
depending on the strength of the fluorescent reporter
molecule (Madisen et al. 2010). For this reason, most
labs wait 5–10 days after tamoxifen injection to
analyze reporter samples.
Cre/CreER LINES FOR THE INNER EAR
In recent years, there has been a significant expansion
of Cre and CreER mouse lines that are useful for inner
ear research. We will continue to have additional strains
in the future due to the NIH Neuroscience Blueprint
Cre-Driver Network whose goal is to provide the
Neuroscience Community with mouse strains suitable
for the cell type-specific perturbation of gene function
in the nervous system (http://www.credrivermice.org/
index). In addition, the NIH Knockout Mouse Project
(KOMP) and the European Conditional Mouse Mutant
Program have initiated plans to create floxed alleles for
every gene in the mouse genome (Austin et al. 2004;
Auwerxetal.2004).Thecombinationoftheseresources
offers the unique opportunity to study the role of any
GOI in a particular cell type of the inner ear at a
particular age. Here, we discuss conditional gene
expression relevant to research in the inner ear with a
review of Cre and CreER mouse lines that have Cre
activity in specific cell types (Tables 1 and 2), as well as a
discussion of practical issues that arise when using
conditional gene expression. In the following
section, several Cre/CreER alleles are presented
more than once as their Cre expression pattern
has been described in multiple inner ear cell types
and at different stages of development. It is
important to note that the majority of the Cre/
CreER alleles that we discuss have Cre expression
in other tissues and organs in addition to the inner
ear.
Cre/CreER LINES FOR THE DEVELOPING OTIC
VESICLE AND OTOCYST
This section will summarize mouse lines with Cre
expression occurring in the developing structures of
the inner ear during early embryogenesis. While
reporter activity persists to postnatal ages, the onset
of Cre expression began embryonically.
Foxg1-Cre is a knock-in mouse line that is a
modified version of the Foxg1-lacZ mouse (Xuan et
al. 1995) where the Cre coding sequence was inserted
into the Foxg1 locus and disrupts endogenous Foxg1
expression. Using ROSA26LacZreporter mice (Hebert
and McConnell 2000) and Z/AP reporter mice
(Pirvola et al. 2002), Cre+cells were first detected at
embryonic day (E)8.5 in the otic placode. By E13.5,
reporter expression was found throughout the otic
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Page 6

TABLE 1
Summary of Cre lines described in the review
Conditional allele
Type
Expression pattern
Level of expression
Original citation
Source
Atoh1-Cre
Knock-in
E13.5: vestibular sensory regions; E14.5: basal
turn of the cochlea; P0: HCs and SCs in
cochlea and vestibular organs
Cochlear and vestibular
HCs (90 %), cochlear
SCs (60 %), utricle SCs
(6 %), saccule SCs (15 %), cristae SCs (42 %)
Yang et al. (2010a)
Not commercially
available
Atoh1-Cre
Transgenic
E11: otocyst; E18.5: HCs and SCs in cochlea
and vestibular organs, spiral and vestibularganglion neurons
Cre expression not
quantified
Matei et al. (2005)
Jax stock #11104
CAG-Cre
Transgenic
Ubiquitous expression
Cre expression not
quantified
Sakai and Miyazaki
(1997)
Not commercially
available
Col1A1-Cre
Transgenic
Not characterized with a reporter line
Liu et al. (2004)
MMRRC stock
#15398-UCD
Col2A1-Cre
Transgenic
E10.5: non-sensory regions of the otocyst and
mesenchymal cells
Cre expression not
quantified
Ovchinnikov et al.
(2000)
Jax stock #3554
Fgf16-Cre
Knock-in
E10.5: otic vesicle; P1: semicircular canal cristae, stria vascularis, cochlear spiral
prominence epithelium
Cre expression not
quantified
Hatch et al. (2009)
Not commercially
available
Foxg1-Cre
Knock-in
E8.5: otic placode; E13.5: cochlea, vestibular
organs, spiral and vestibular ganglia
Cre expression not
quantified
Hebert and McConnell
(2000)
Jax stock #4337
hGFAP-Cre
Transgenic
E13.5: utricle, cristae, non-sensory cells around
vestibular organs, postnatal cochlear SCs
Cre expression not
quantified
Zhuo et al. (2001)
Jax stock #4600
Gfi1-Cre
Knock-in
E13.5: vestibular HCs; E15.5: cochlear HCs in
basal turn; E18.5: cochlear and vestibular HCs
Cochlear HCs (93 %),
HCs in
utricle, saccule, and
cristae (90 %)
Yang et al. (2010b)
Not commercially
available
Hoxb2-r4-Cre
Transgenic
E8.5: otic placode; P0: cochlea, vestibular
organs, spiral and vestibular ganglia
Cre expression not
quantified
Szeto et al. (2009)
Not commercially
available
Otog-Cre
Transgenic
Not characterized with a reporter line
Cohen-Salmon et al.
(2002)
Not commercially
available
Pax2-Cre
BAC
transgenic
E8.5: otic placode; P0: cochlea, vestibular
organs, spiral and vestibular ganglia
Cre expression not
quantified
Ohyama and Groves
(2004)
MMRRC stock
#10569-UNC
Pax3-Cre
Knock-in
E9: otic vesicle and developing spiral and
vestibular ganglion; E11.5 to E17.5: cochlea,
utricle and saccule
Cre expression not
quantified
Engleka et al. (2005)
Jax stock #5549
Pax8-Cre
Knock-in
E8.5: otic placode; E16.5: epithelial components
of the inner ear and spiral and vestibular ganglia
Cre expression not
quantified
Bouchard et al. (2002)
EMMA stock #EM:
00141
Pou3f4-Cre
Transgenic
E14.5: otic mesenchyme, adult: temporal bone,
spiral ligament, spiral limbus, tympanic border
cells, Reissner’s membrane, mesenchymal cells,
non-sensory cells in utricle
Cre expression not
quantified
Ahn et al. (2009)
Not commercially
available
Pou4f3-Cre
Transgenic
E12.5: utricle sensory epithelium; E13.5: zone
of non-proliferating cells in cochlea; P6:
cochlear HCs and SCs, vestibular HCs and
stroma
Cre expression not
quantified
Sage et al. (2006)
Not commercially
available
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Page 7

epithelium, including the cochlea, vestibular organs,
and both spiral and vestibular ganglia, while the
surrounding mesenchyme was Cre-negative. Foxg1-
Cre has been used in numerous studies to investigate
the deletion or overexpression of various genes
(Pirvola et al. 2002; Arnold et al. 2006; Zelarayan et
al. 2007; Barrionuevo et al. 2008; Jones et al. 2008;
Rickheit et al. 2008; Grimsley-Myers et al. 2009;
Schultz et al. 2009; Wang et al. 2009; Yamamoto et
al. 2009; Deng et al. 2010; Freyer and Morrow 2010;
Haugas et al. 2010; Hurd et al. 2010; Hwang et al.
2010; Sipe and Lu 2011). We highlight a few studies
here. Yamamoto et al. (2011) challenged the estab-
lished dogma that Notch signaling is required to
specify cochlear sensory progenitor cells using Foxg1-
Cre-mediated deletion of RBP-J. Three other papers
used Foxg1-Cre to delete Notch receptors (Delta1,
Jagged1, and Notch1) to demonstrate a role for Notch
signaling in lateral induction and lateral inhibition in
the inner ear (Kiernan et al. 2005, 2006; Brooker et al.
2006). Foxg1-Cre was also used to ectopically express
the Notch intracellular domain (NICD) which showed
that Notch signaling is sufficient to generate ectopic
sensory patches (Hartman et al. 2010; Pan et al. 2010).
The role of N-myc in the development of the otic
vesicle and its control over proliferation in the otic
epithelium were discovered using both Foxg1-Cre and
Pax2-Cre lines (Dominguez-Frutos et al. 2011;
Kopecky et al. 2011). Comparison of the germline
knockout of Sonic hedgehog (SHH) with Foxg1-Cre-
mediated deletion of Smoothened demonstrated
SHH’s direct role in the formation of ventral otic
structures (cochlea and saccule) and SHH’s indirect
role in the formation of dorsal structures (utricle,
semicircular canal cristae, and the endolymphatic
duct). Conditional deletion of Smoothened also
revealed that SHH signaling regulates the prolifera-
tion of neurogenic progenitors which give rise to
spiral and vestibular ganglion neurons (Brown and
Epstein 2011). Fgf8’s role in regulating the develop-
ment of cochlear pillar cells was also discovered using
the Foxg1-Cre allele (Jacques et al. 2007). Heterozy-
gous Foxg1-Cre mice have only one copy of the Foxg1
gene (Hebert and McConnell 2000), which has been
reported to cause haploinsufficiency phenotypes that
include proliferation in other organs (Shen et al.
2006; Eagleson et al. 2007; Siegenthaler et al. 2008).
However, no change in proliferation in the inner ear
has been reported in several papers where proper
controls of Foxg1-Cre mice (without the floxed allele)
were used (Yamamoto et al. 2009, 2011; Hartman et al.
2010; Brown and Epstein 2011).
The Pax2-Cre transgenic allele was created using a
BAC that contains a 101-kb region 5′ to the mouse
Pax2 gene as well as 20 kb of the Pax2 coding region,
which includes the first three exons of the Pax2 gene.
Prestin-Cre
BAC transgenic
P6: cochlear and vestibular HCs, spiral ganglia
region
Cre expression not
quantified
Tian et al. (2004)
Not commercially
available
Prestin-Cre
Transgenic
P14: cochlear inner HCs, vestibular HCs, spiral
ganglia region; P50: last row of cochlear outer HCs
Cre expression not
quantified
Li et al. (2004)
Not commercially
available
Prox1-eGFP/Cre
Knock-in
P23: cochlear HCs, pillar cells, Deiters’ cells, GER
and LER
Outer HCs (30 %),
inner HCs (4 %),
pillar cells and
Deiters’ cells (almost
100 %)
Liu et al. (2012)
Not commercially
available
SHH-EGFP/Cre
Knock-in
P0: spiral ganglion neurons, peripheral nerve fibers
surrounding HCs and SCs
Spiral ganglion
neurons (100 %)
Harfe et al. (2004)
Jax stock #5622
Sox2-Cre
Transgenic
Ubiquitous expression
Cre expression not
quantified
Hayashi et al.
(2002)
Jax stock #4783
Wnt1-Cre
Transgenic
E9: otic vesicle, developing spiral and vestibular ganglion; E11.5 to E17.5: cochlea, utricle and saccule
Cre expression not
quantified
Danielian et al.
(1998)
Jax stock #3829
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Page 8

TABLE 2
Summary of CreER lines described in the review
Conditional allele
Type
Tamoxifen induction
Expression pattern
Original citation
Source
Atoh1-CreERTM
Transgenic
E16 only
Cochlear HCs (not quantified)
Chow et al. (2006)
MMRRC stock
#29581-UNC
P0 only
Cochlear inner HCs (40 %), outer HCs (50 %), utricle and
saccule HCs (40 %), cristae HCs (10 %)
P0 and P1
Cochlear inner HCs (80 %), outer HCs (90 %), utricle and
saccule HCs (60 %), cristae HCs (15 %)
P0, P1, and P2
Cochlear inner HCs (80 %), outer HCs (90 %), utricle and
saccule HCs (60 %), cristae HCs (20 %)
Atoh1-CreERT2
Transgenic
E12.75
Cochlear inner HCs (40 %), outer HCs (44 %)
Machold and Fishell
(2005)
Jax stock #7684
E12.75 and E13.75
Cochlear inner HCs (90 %), outer HCs (97 %)
P0 and P1
Cochlear HCs (10–20 % depending on turn), utricle HCs
(not quantified)
CAG-CreERTM
Transgenic
Varies
Ubiquitous expression
Hayashi and McMahon
(2002)
Jax stock #4453
Fgfr3-iCreERT2
PAC transgenic
P0 and P1
Pillar and Deiters’ cells (100 %), cochlear outer HCs (25–
75 % depending on turn), Hensen or Claudius cells (not quantified), spiral lamina
Rivers et al. (2008)
Not commercially
available
P2 and P3
Pillar and Deiters’ cells (100 %), cochlear outer HCs (930
% depending on turn), Hensen or Claudius cells (not
quantified), spiral lamina
P6 and P7
Pillar and Deiters’ cells (100 %), cochlear outer HCs (920 %
depending on turn), Hensen or Claudius cells (not quantified),
spiral lamina
P12 and P13 or P30
Pillar and Deiters’ cells (100 %), Hensen or Claudius cells (not
quantified), spiral lamina
Ngn1-CreERT2
BAC transgenic
E8.5 to E13.5
HCs, SCs and non-sensory epithelium of vestibular organs,
spiral and vestibular ganglia, GER (no quantifications were done)
Raft et al. (2007)
Jax stock #8529
E8.5 only
Vestibular ganglion neurons (not quantified)
E12.5 only
Spiral ganglion neurons (not quantified)
Plp-CreERT2
Transgenic
P0–P7, P3–P9, or
P10-P16
Inner phalangeal cells, Schwann cells in the spiral lamina,
vestibular SCs, vestibular Schwann cells and satellite cells (no
quantifications were done)
Doerflinger et al.,
(2003)
Jax stock #5975
P0 and P1
Inner phalangeal cells (50–80 % depending on turn), pillar and
Deiters’ cells (5–10 %), utricle HCs and SCs (not quantified)
Prestin-CreERT2
Knock-in
P0, P1, and P2
Cochlear outer HCs (15–60 % depending on turn)
Fang et al. (2012)
Not commercially
available
P2 and older
Cochlear outer HCs (100 %)
Prox1-CreERT2
Knock-in
E16
Deiters’ cells (72 %), outer pillar cells (18 %), inner pillar cells
(3 %), cochlear outer HCs (7 %)
Srinivasan et al.
(2007)
Not commercially
available
P0 and P1
Pillar cells (5 %), Deiters’ cells (5–10 %)
ROSA26-CreER
Knock-in
Varies
Ubiquitous expression
Vooijs et al. (2001)
Not commercially
available
ROSA26-CreER
Knock-in
Varies
Ubiquitous expression
Badea et al. (2003)
Jax stock #4847
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Page 9

An IRES followed by the Cre coding sequence was
inserted into exon 2 of this Pax2 sequence. Cre
expression patterns were characterized with ROSA26-
LacZand Z/EG reporter mice and first detected at
E8.5 in the otic placode. By postnatal day (P)0, most
cells in the cochlea and vestibular organs were Cre+,
including cells in the organ of Corti, stria vascularis,
Reissner’s membrane, sensory epithelia of the vestib-
ular organs, and both the spiral and vestibular ganglia
(Ohyama and Groves 2004). Pax2-Cre has also been
used extensively (Arnold et al. 2006; Rocha-Sanchez et
al. 2007; Doetzlhofer et al. 2009; Grimsley-Myers et al.
2009; Soukup et al. 2009; Wang et al. 2009; Abraira et
al. 2010; Fritzsch et al. 2010; Jahan et al. 2010a; Basch
et al. 2011; Dominguez-Frutos et al. 2011; Kopecky et
al. 2011; Pan et al. 2011). Neurod1 deletion using
Pax2-Cre disrupted the basal-to-apical gradient of hair
cell (HC) differentiation and produced ectopic HCs
in the spiral and vestibular ganglia regions. These
results suggest an antagonistic relationship between
Neurod1 and Atoh1 during inner ear development
(Jahan et al. 2010b). Pax2-Cre was also used to
demonstrate that bone morphogenetic protein
(BMP) signaling promotes the formation of the
abneural side of the cochlea (i.e., organ of Corti and
lesser epithelial ridge (LER)) while repressing the
formation of the neural side (i.e., greater epithelial
ridge (GER); Ohyama et al. 2010). Pax2-Cre-mediated
deletion and activation of canonical Wnt signaling
demonstrated Wnt’s role in mediating the otic
placode–cranial epidermis fate decision by promoting
an otic placode fate (Ohyama et al. 2006). The Pax2-
Cre allele was also used to show the interaction
between Notch signaling and Wnt signaling in the
otic placode (Jayasena et al. 2008).
Pax3-Cre is a knock-in mouse line where the Cre
coding sequence was inserted into the first exon of
the Pax3 gene, creating a null allele of Pax3.
Homozygous Pax3-Cre mice die embryonically
(Engleka et al. 2005). Cre expression was charac-
terized in the developing otic epithelium using the
RCE:loxP reporter where the CAG promoter was
inserted after the ROSA26 locus followed by a
floxed “stop” sequence and eGFP (Sousa et al.
2009). Cre+cells were first detected at E9 in the
prosensory epithelium of the otic vesicle, as well as
within the developing spiral and vestibular ganglion.
In later stages of development (E11.5 to E17.5), Pax3-
Cre activity is present in HCs and supporting cells
(SCs) of the cochlea, utricle, and saccule. There was
also Cre activity detected in the cells of the GER, stria
vascularis, endolymphatic duct, and periotic mesen-
chyme. No Cre activity was detected in the semicircu-
lar canals cristae at any age. This use of Pax3-Cre
(together with Wnt1-Cre described below) provided
the novel and unexpected finding that embryonic
neural tube cells contribute to the formation of the
otic vesicle and can develop into both sensory and
non-sensory cells (Freyer et al. 2011).
The transgenic Wnt1-Cre line uses the Wnt1
enhancer to drive the expression of Cre (Danielian
et al. 1998). Using the RCE:loxP reporter, Cre
expression was very similar to the Pax3-Cre line, but
with fewer Cre+cells in each of the locations (Freyer
et al. 2011).
Pax8-Cre is a knock-in mouse line where the Cre
coding sequence replaced exon 3 of the Pax8 gene,
creating a null allele of Pax8 (Bouchard et al. 2002).
Using the Z/AP reporter line, Cre activity was first
detected at E8.5 in the otic placode, and by E10.5,
most cells in the otic vesicle were Cre+. At E16.5, most
epithelial components of the inner ear had Cre
activity, as well as the spiral and vestibular ganglia;
however, reporter expression in the semicircular
canal cristae was patchy (Bouchard et al. 2004).
The transgenic Hoxb2-r4-Cre line uses the Hoxb2
r4 enhancer, a 1.4-kb fragment of the 5′ region of the
Hoxb2 gene, to drive the expression of Cre. Using the
ROSA26LacZreporter line, βgal expression was first
detected in the pre-otic field at the five-somite stage
(~E8.5); thus, when the otic placode and otic vesicle
develop, these cells are Cre+as well. Accordingly, at
P0, most cells are Cre+in the cochlea and vestibular
organs, as well as in the spiral and vestibular ganglia,
stria vascularis, and Reissner’s membrane (Szeto et al.
2009).
There are several Cre lines that use a collagen
promoter to drive the expression of Cre recombinase.
Col1A1-Cre is a transgenic line that uses a 3.6-kb
fragment of the rat alpha1(I) collagen (Col1A1)
promoter (Liu et al. 2004). This line was not
characterized in the developing otocyst using a
reporter allele; however, endogenous Col1A1 was
detected by in situ hybridization at E11.5 with
ubiquitous expression in the otocyst. Col1A1-Cre was
used to delete the retinoblastoma protein (Rb) in the
developing inner ear, which resulted in an increase in
HCs and SCs in both the cochlea and utricle. Rb-null
HCs had stereocilia bundles, were innervated, and
continued to divide at late embryonic ages. Thus, cell
fate determination and differentiation into HCs and
SCs does not require the presence of Rb and can
occur even while cells are proliferating (Sage et al.
2005). There is a second Col1A1-Cre allele that uses a
truncated 2.3-kb fragment of the rat Col1A1 promoter
(Liu et al. 2004), but this line has not been described
in the inner ear.
The type II collagen promoter (Col2A1) has also
been used to generate a transgenic Cre mouse line.
Col2A1-Cre uses 3 kb of the Col2A1 promoter, the first
exon of Col2A1, with a mutated initiation codon, and
a 3.02-kb fragment of intron 1 followed by IRES to
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Page 10

drive the expression of Cre (Ovchinnikov et al. 2000).
Using the ROSA26LacZreporter line, βgal expression
was first detected at E10.5 as small clusters of cells in
the non-sensory regions of the otocyst, as well as in
surrounding mesenchymal cells. Col2A1-Cre has been
used to ectopically express the NICD in a transient
manner by combining it with the tetracycline-induc-
ible system. The expression of NICD induced ectopic
HCs and SCs in the non-sensory regions of the
cochlea and vestibular system, demonstrating that
Notch signaling is sufficient for the initiation of
sensory cell fate in the developing inner ear (Pan et
al. 2010).
Otogelin-Cre or Otog-Cre is a transgenic BAC
mouse line where Cre is under the control of the
murine Otog promoter. A reporter line was not used to
characterize Otog-Cre; however, endogenous Otog
expression was detected at E10 in the otic vesicle
and at E18 in the non-sensory cells of the sensory
epithelium of the cochlea and vestibular organs.
Otog-Cre-mediated deletion of connexin26 resulted
in normal development of the inner ear, but by
hearing onset, progressive cell death was described
beginning with inner phalangeal and border cells and
continuing to outer HCs and other SC subtypes. Inner
HCs remained intact and the vestibular system was not
affected. These results demonstrate that connexin26
and the gap junction network are required for the
survival of cochlear HCs and SCs and, thus, auditory
function (Cohen-Salmon et al. 2002).
The BAC transgenic line, Neurogenin1-CreERT2
(also called Ngn1-CreERT2or Neurog1-CreERT2),
contains 113 kb of the 5′ sequence, the Ngn1 coding
sequence, and 71 kb of the 3′ sequence. The CreERT2
coding sequence replaced the Ngn1 coding sequence
in the BAC. Using the Z/EG reporter line and
tamoxifen (1 mg/40 g) given twice daily by gavage
from E8.5 to E13.5, analysis at E14.5 revealed many
Cre+HCs and SCs in the utricle and saccule, while
only a few Cre+SCs were found in the cristae. There
were also many Cre+cells in the surrounding non-
sensory epithelium of the utricle and saccule as well as
in the spiral and vestibular ganglia. In the cochlea,
Cre+cells were found only in the GER. Similar results
were described when tamoxifen was given at E8.5 and
E9.5. These results indicate that the sensory regions of
the utricle and saccule, but not the cristae and
cochlea, are derived from the neurogenic region of
the otocyst (Raft et al. 2007). More fate mapping
studies using the Ngn1-CreERT2line were conducted
to determine the specification of vestibular and spiral
ganglion neurons. Early tamoxifen injections (E8.5)
primarily labeled vestibular ganglion neurons, while
tamoxifen given at E12.5 primarily labeled spiral
ganglion neurons; thus, Ngn1+precursor cells change
with age to generate these two cell populations
(Koundakjian et al. 2007). Ngn1-CreERT2-mediated
deletion of ephrin-B2 with tamoxifen given by gavage
at E9.5–E10.5 demonstrated ephrin-B2’s role in the
fasciculation of spiral ganglion neurons (Coate et al.
2012). There is also a non-inducible Ngn1-Cre allele
that used the same BAC construct, but it has only
been described in the brain (Lundell et al. 2009).
Fgf16-Cre is a knock-in mouse line with an IRES-
Cre cassette inserted into the first coding exon of
Fgf16. Even though this line is a null allele, the inner
ears of homozygotes are structurally and functionally
normal. Using the ROSA26LacZreporter line, βgal
expression was first apparent in the otic vesicle at
E10.5. By P1, Cre+cells were detected in the sensory
and non-sensory regions of the semicircular canal
cristae, at the base of the stria vascularis, and in the
cochlear spiral prominence epithelium (Hatch et al.
2009).
Cre/CreER LINES FOR THE DEVELOPING
ORGAN OF CORTI
This section will focus on Cre lines with expression
occurring in the developing sensory epithelium of the
cochlea (Fig. 2) during late embryogenesis, when
prosensory cells are in the process of committing to
either a HC or SC fate.
Pou4f3-Cre is a transgenic allele which uses a 9-kb
region 5′ to the Pou4f3 gene as the promoter to drive
Cre expression. Using the ROSA26LacZreporter, βgal
expression was first detected at E13.5 in the zone of
non-proliferating cells of the developing cochlea and
by P6 in both HCs and SCs. Quantification of Cre+
cells was not reported (Sage et al. 2006). Pou4f3-Cre
was used to delete Rb, which extended HC and SC
proliferation to neonatal ages while preserving the
initial development of mechanoelectrical transduc-
tion; however, degeneration occurred in the adult
organ of Corti that led to severe hearing loss. These
experiments suggest that transient regulation of Rb
might be a strategy to achieve cochlear HC regener-
ation (Sage et al. 2006). Pou4f3-Cre-mediated dele-
tion of Dicer1 caused malformations in HC stereocilia
bundles, degeneration of HCs, and subsequent hear-
ing loss. Interestingly, vestibular HCs were less
effected and only a mild vestibular phenotype was
detected (Friedman et al. 2009) The Pou4f3-Cre line
has also been used to ablate HCs in a mosaic but
reproducible manner by the expression of a drug-
inducible, dimerizable caspase 3 (iCsp3) that leads to
hearing loss after a week of drug administration. In
the cochlea, ~60 % of HCs expressed iCsp3 (Fujioka
et al. 2011).
There are two Atoh1-Cre lines, a knock-in allele
(Yang et al. 2010a) and a transgenic allele (Matei et al.
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Page 11

2005). The Atoh1-Cre knock-in line replaced the
entire Atoh1 coding sequence with the Cre coding
sequence. Homozygous Atoh1-Cre mice die soon after
birth, whereas heterozygous Atoh1-Cre mice are
viable and display no visible defects. Using the
ROSA26LacZreporter, βgal expression was first
detected in the basal turn of the cochlea at E14.5
and progressed to middle and apical turns over the
next few days. By P0, βgal expression was observed in
all turns of the cochlea in both HCs and SC, including
Deiters’ cells, pillar cells, and inner phalangeal cells.
Approximately 90 % of HCs and 60 % of SCs were
Cre+. These findings prompted the conclusion that
Atoh1 is expressed in prosensory progenitor cells
before HC and SC fates are specified (Yang et al.
2010a). The Atoh1-Cre transgenic line used the same
Atoh1 enhancer fragment used to create the Atoh1-
eGFP mouse (Chen et al. 2002) and was also
characterized with the ROSA26LacZreporter line. In
contrast to the Atoh1-Cre knock-in line, βgal expres-
sion was first detected in the Atoh1-Cre transgenic
line at E11 in areas that correspond to the future
sensory epithelium. At E18.5, almost all cochlear HCs
expressed βgal, except for those in the most apical
tip. The Atoh1-Cre transgenic line also had Cre
expression in SCs. Quantification of Cre+cells was
not reported (Matei et al. 2005). The Atoh1-Cre
transgenic line has been used to delete Dicer1,
showing the important roles of microRNAs in cochle-
ar gene expression profiles and maintaining the apex
to base gradient of gene expression. This model also
demonstrated that microRNAs are required for HC
survival (Weston et al. 2011). Transgenic Atoh1-Cre-
mediated deletion of beta or gamma actin isoforms
showed that only one of these genes is needed for the
normal development of HC stereocilia bundles, while
each gene plays a different role in the maintenance of
stereocilia during aging (Perrin et al. 2010). Compar-
ison of Atoh1-Cre-mediated Neuro1d deletion with
Pax2-Cre-mediated Neuro1d deletion demonstrated
that Neurod1’s roles in HC differentiation and
maturation are relevant only at very early embryonic
ages (Jahan et al. 2010b).
There are also two inducible Cre alleles which use
the Atoh1 enhancer to drive the expression of CreER:
Atoh1-CreERTM(Chow et al. 2006) and Atoh1-
CreERT2(Machold and Fishell 2005). The transgenic
Atoh1-CreERTMallele was induced with tamoxifen
(100 μg/g, IP) at E16 and analyzed at E19 using a
Rosa26-loxP-stop-loxP-NICD-IRES-eGFP line (which
expresses both NICD and eGFP after Cre-mediated
excision of the floxed stop sequence). eGFP expres-
sion was only detected in HCs; however, quantifica-
tion was not performed. The eGFP+HCs in this model
also had ectopic expression of NICD, but appeared
normal (Liu et al. 2012b). The transgenic Atoh1-
CreERT2line was characterized with the ROSA26eYFP
reporter and embryonic tamoxifen induction (the
tamoxifen dose and route of administration were not
specified). After a single tamoxifen dose at E12.75,
eYFP expression was detected in 40 % of inner HCs
and 44 % of outer HCs at E18.5. These percentages
increased to 90 % of inner HCs and 97 % of outer
HCs when a second tamoxifen dose was given at
FIG. 2.
epithelium of the cochlea and utricle. In the magnified image of the
utricle, HCs are in pink and SCs are in blue. The magnified image of
the cochlea focuses on the organ of Corti, with HCs in pink. Green
Diagram of the inner ear and cell types in the sensory
SCs underneath the inner HC are inner phalangeal cells/border cells.
Brown SCs between the inner and outer HCs are pillar cells. Yellow
SCs underneath the three outer HCs are Deiters’ cells. The orange SC
lateral to the outer HCs is a Hensen cell.
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Page 12

E13.75. Cre activity was not observed in SCs with
either induction paradigm. The Atoh1-CreERT2allele
was used to delete Eya1 and Six1, demonstrating that
these genes are required for HC development and
Atoh1 expression (Ahmed et al. 2012).
Gfi1-Cre is a knock-in allele where the Cre coding
sequence replaced the endogenous Gfi1 gene; thus,
homozygous Gfi1-Cre mice cannot be used because of
their severe phenotype where HCs do not form. This
line was characterized with the ROSA26LacZreporter
where βgal expression was first seen in inner HCs of
the basal turn at E15.5 that progressed to outer HCs
and other turns of the cochlea over the following
days. By E18.5, nearly all HCs (~93 %) in the entire
cochlea were labeled by βgal. No other cell types in
the cochlea were labeled (Yang et al. 2010b). Gfi1-Cre-
mediated deletion of BMP2 demonstrated that this
gene is not required for normal HC formation or
hearing ability (Hwang et al. 2010).
The Prox1-CreERT2knock-in allele was created by
inserting IRES-CreERT2into the mouse Prox1 locus
(Srinivasan et al. 2007). Heterozygous Prox1-CreERT2
mice have normal Prox1 expression, normal mor-
phology of the organ of Corti, and normal hearing.
Homozygous mice were not used since the Cre coding
sequence is followed by a Neo-cassette which may
affect the expression levels of Prox1 (Yu et al. 2010).
The Cre expression pattern was characterized using
the ROSA26eYFPreporter line and tamoxifen (100 μg/
g, IP) given at E16. When the cochlea was analyzed at
E19, Cre expression was detected in ~72 % of Deiters’
cells, ~18 % outer pillar cells, ~3 % inner pillar cells,
and ~7 % of outer HCs. No Cre+inner HCs were
found (Liu et al. 2012b).
Cre/CreER LINES FOR THE DEVELOPING
VESTIBULAR ORGANS
This section will focus on Cre lines with expression
occurring in the developing sensory epithelium of
vestibular organs: the utricle, saccule, and semicircu-
lar canal cristae (Fig. 2).
Cre activity from the transgenic Pou4f3-Cre mouse
line was first seen using the ROSA26LacZreporter in
the sensory epithelium of the utricle at E12.5. By
E13.5, βgal expression was detected in vestibular HCs.
At P6, the only βgal+cells in the sensory epithelium
were HCs, but there were also βgal+cells in the
stroma (containing nerve fibers) beneath the epithe-
lium. Quantification of Cre+cells was not reported.
Pou4f3-Cre-mediated deletion of Rb was also studied
in the utricle where HCs continued to divide until
6 weeks of age and partial vestibular function was
maintained at 6 months. Vestibular Rb-null HCs died
at a slow rate, which contrasts with the rapid cell death
seen in cochlear HCs when Rb was deleted using the
same Cre line. Thus, Rb plays different roles in the
survival of these two types of HCs (Sage et al. 2006).
ROSA26LacZreporter expression was also detected
in the vestibular system with the two Atoh1-Cre lines.
Cre+cells in the Atoh1-Cre knock-in allele were first
detected in vestibular sensory regions at E13.5 and
increased to P0 where both HCs and SCs in the
utricle, saccule, and cristae expressed βgal. Specifical-
ly, there were ~90 % Cre+HCs in each of these
organs, and the number of Cre+SCs varied from 6 %
in the utricle to 15 % in the saccule and 42 % in the
cristae. These data also suggest that, as in the cochlea,
Atoh1 is expressed in vestibular progenitor cells
before HC and SC fates are specified (Yang et al.
2010a). Cre activity in vestibular organs of the Atoh1-
Cre transgenic line was detected at E11 and by E18.5
was found in vestibular HCs and SCs. Quantification
of Cre+cells was not reported (Matei et al. 2005). The
Atoh1-CreERTMand Atoh1-CreERT2alleles were not
characterized in the embryonic vestibular system.
The knock-in Gfi1-Cre allele also has Cre expres-
sion in vestibular HCs beginning at E13.5 and
progressively increasing to P0, where ~90 % of HCs
in the saccule, utricle, and cristae are βgal+. No
vestibular SCs were Cre+(Yang et al. 2010b).
Cre/CreER LINES FOR THE POSTNATAL ORGAN
OF CORTI
This section will focus on Cre lines with expression
occurring in the sensory epithelium of the cochlea
after birth.
The transgenic Atoh1-CreERTMline also has Cre
activity in the postnatal cochlea After tamoxifen
injection(s) (3 mg/40 g, IP) at birth, the only Cre+
cells found in the organ of Corti were HCs (as
revealed using the ROSA26LacZreporter line). One
tamoxifen injection at P0 resulted in 40 % Cre+inner
HCs and 50 % Cre+outer HCs. These percentages
increased to 80 % Cre+inner HCs and 90 % Cre+
outer HCs when tamoxifen was given once at P0 and
again 24 h later at P1. No further increase was seen
with a third injection given at P2 (Chow et al. 2006;
Weber et al. 2008). In contrast to the results obtained
with embryonic Rb deletion using the Col1A1-Cre
(Sage et al. 2005) and Pou4f3-Cre lines (Sage et al.
2006), deletion of Rb in neonatal HCs using Atoh1-
CreERTMproduced S phase reentry followed by cell
death. Thus, Rb plays an age-dependent role in HC
proliferation (Weber et al. 2008). Atoh1-CreERTM-
mediated deletion of Pkd1 ruled out the involvement
of this protein as the major component in the
mechanoelectrical transduction channel complex or
in planar cell polarity mechanisms, but demonstrated
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Page 13

its requirement for normal stereocilia number and
structure (Steigelman et al. 2011). This line has also
been used to drive the expression of DTA as a method
of damaging HCs in the neonatal cochlea, in vivo
(Cox et al. 2010). Ectopic NICD expression in
neonatal HCs using the Atoh1-CreERTMallele
resulted in the reactivation of Sox2 in inner and
outer HCs and of Prox1 only in outer HCs. Despite
the expression of NICD, Sox2, and Prox1, HCs
continued to develop normally, demonstrating that
once a HC fate is committed activation of the Notch
pathway will not impact their development (Liu et al.
2012b).
We also investigated the Cre expression pattern of
Atoh1-CreERT2mice (Machold and Fishell 2005) with
tamoxifen induction after birth. We bred this line with
the ROSA26eYFPreporter and gave tamoxifen injec-
tions (3 mg/40 g, IP) once a day at P0 and P1.
Analysis at P6 revealed only Cre+HCs, but a much
lower number than what was observed with the Atoh1-
CreERTMline under the identical conditions. We
found ~20 % Cre+inner and outer HCs in the apical
turn of the cochlea and G10 % in middle and basal
turns (Fig. 3). Both Atoh1-CreER strains are trans-
genics; thus, positional effects and the copy number
of the transgene likely play a role in the difference of
Cre activity observed.
There are three mouse strains that use some form
of the prestin locus to drive the expression of Cre or
CreER: Prestin-Cre (transgenic), Prestin-Cre (BAC
transgenic), and Prestin-CreERT2. The Prestin-Cre
(transgenic) allele uses a 9-kb fragment of mouse
prestin, which contains the putative thyroid hormone-
responsive element, the first coding exon (exon 3),
and part of intron 3, but not the promoter region
upstream of exon 1, to drive the expression of Cre.
Using the ROSA26LacZreporter line, Cre activity was
first detected at P14 in inner HCs, and this increased
so that by P135 most inner HCs were Cre+. In
addition, faint βgal expression was observed in the
last row of outer HCs in the apical and middle turns
of the cochlea starting at P50 (Li et al. 2004).
However, analysis of successive generations of this
line with the ROSA26eYFPand Ai6 (where deletion of
the floxed “stop” sequence drives the expression of
ZsGreen; Madisen et al. 2010) reporter lines did not
reveal detectable reporter signals in adult HCs, likely
due to genetic drift of transgenic expression (Dear-
man and Zuo, unpublished).
The Prestin-Cre (BAC transgenic) mouse line used
a modified BAC containing ~150 kb of genomic DNA
which includes the coding region of prestin (~50 kb)
followed by an IRES-Cre cassette after the stop codon
in exon 20. This line has only a single copy of the
transgenic BAC; however, it was not determined how
much of the prestin promoter region is contained
within the BAC, which may be the reason why Cre
expression is different from endogenous prestin
expression. When this line was characterized with
the ROSA26LacZreporter line, βgal expression was
first detected at P6 in the majority of outer HCs in the
middle and basal turns of the cochlea, while only a
few outer HCs in the apical turn were Cre+. Some
inner HCs also had βgal expression, and this expres-
sion level increased between P6 and P60 (Tian et al.
2004). Conditional deletion of the adherence junc-
tion protein, vezatin, using the Prestin-Cre (BAC
transgenic) line showed no effects on hearing devel-
opment or function at early ages, but an increased
sensitivity to noise and age-related hearing loss. These
findings highlight the role of HC–SC junctions in
hearing (Bahloul et al. 2009). Deletion of thyroid
hormone receptor beta using this Cre line clarified
that malformation of the tectorial membrane (cochle-
ar cause) and not delayed BK channel expression in
HCs (retro-cochlear cause) produced hearing loss in
the absence of this receptor (Winter et al. 2009).
Most recently, Prestin-CreERT2was generated us-
ing a knock-in method which resulted in a Cre
expression pattern that more accurately recapitulates
the endogenous expression of prestin. IRES-CreERT2
was inserted into the prestin locus after the stop codon
in exon 20. Unlike most knock-in alleles, endogenous
prestin was not affected and homozygous Prestin-
CreERT2mice have normal hearing, as tested by
auditory brainstem response (ABR). Using the
CAGeGFPand Ai6 (where deletion of the floxed “stop”
sequence drives expression of ZsGreen; Madisen et al.
2010) reporters, Cre expression patterns were investi-
gated after different tamoxifen induction paradigms.
Cre activity was very specific to the outer HCs of the
cochlea with all induction paradigms. Early tamoxifen
injections (3 mg/40 g, IP) once a day at P0, P1, and
P2 produced a gradient of Cre+outer HCs with 60 %
in the base, 35 % in the middle, and 15 % in the apex.
In contrast, tamoxifen injections given once a day for
2 days beginning at P2 or any time after resulted in
close to a 100 % of Cre+outer HCs throughout the
length of the cochlea (Fang et al. 2012).
The Prox1-CreERT2knock-in allele (Srinivasan et
al. 2007) also has Cre expression in the postnatal
cochlea. Using several reporter lines and tamoxifen
(3 mg/40 g, IP) injections given once a day at P0 and
P1, only pillar cells and Deiters’ cells were Cre+in the
organ of Corti. Specifically, 5 % of pillar cells were
Cre+throughout the length of the cochlea, and the
percentage of Cre+Deiters’ cells varied among turns
with 10 % in the apex and 5 % in the middle and
base. Deletion of Rb using the Prox1-CreERT2allele
resulted in cell cycle reentry of both pillar cells and
Deiters’ cells, while only pillar cells were able to
complete the cell cycle and increase in number. This
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Page 14

finding highlights the heterogeneity in the role of Rb
between these two SC subtypes (Yu et al. 2010).
There is a second allele which uses the Prox1 locus
to drive Cre expression. Prox1-eGFP/Cre is a knock-
in line where an eGFP/Cre fusion protein was
inserted downstream of the Prox1 translation start
site (Srinivasan et al. 2010). This allele was recently
characterized in the cochlea using the ROSA26eYFP
reporter line. At P23, Cre expression was detected in
almost all pillar cells and Deiters’ cells, ~30 % outer
HCs, and ~4 % inner HCs. There was also Cre activity
detected in the GER and LER (Liu et al. 2012b).
Fgfr3-iCreERT2is a phage artificial chromosome
(PAC) transgenic mouse line that expresses a “codon-
improved” version of Cre (iCre) driven by the Fgfr3
promoter. Specifically, iCreERT2was inserted into the
first exon of the Fgfr3 gene in the PAC allele (Rivers
et al. 2008; Young et al. 2010). Using the Ai14 reporter
line (where deletion of the floxed “stop” sequence
drives expression of tdTomato; Madisen et al. 2010),
we tested tamoxifen induction at several ages where
the majority of Cre+cells in the cochlea were pillar
cells and Deiters’ cells. After tamoxifen (3 mg/40 g,
IP) injection once a day at P0 and P1, tdTomato
expression was detected in ~100 % of pillar and
Deiters’ cells, 25–75 % of outer HCs depending on
the turn of the cochlea, and a small fraction of
Hensen or Claudius cells (Fig. 4A–C). When we gave
tamoxifen (3 mg/40 g, IP) once a day at P2 and P3
(Fig. 4D) or at P6 and P7 (Fig. 4E), the percentage of
Cre+outer HCs decreased to G30 %, while Cre+pillar
and Deiters’ cells remained at 100 %. There were still
some Cre+Hensen or Claudius cells with both
induction paradigms. When we gave tamoxifen
(3 mg/40 g, IP) once a day at P12 and P13 (Fig. 4F)
or one injection of tamoxifen (9 mg/40 g, IP) at P30
FIG. 3.
Atoh1-CreERT2. Representative confocal projection images of the
apical turn of the cochlea (A) and utricle (B) from Atoh1-CreERTM;
ROSA26eYFPmice, induced with tamoxifen at P0 and P1. Repre-
sentative confocal projection images of the apical turn of the cochlea
Comparison of the transgenic lines, Atoh1-CreERTMand
(C) and utricle (D) from Atoh1-CreERT2;ROSA26eYFPmice, induced
with tamoxifen at P0 and P1. In A and C, eYFP is in white. In B and
D, eYFP is in green and the HC marker, myosin VIIa (myo7a), is in
red. Scale bars, 100 μm.
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

Page 15

(data not shown), we no longer observed Cre+outer
HCs, while Cre+pillar and Deiters’ cells remained at
100 %. A low percentage of Cre+Hensen and
Claudius cells also remained. Cre+inner HCs were
never found with any induction paradigm. Cre+cells
were also found in the spiral lamina (data not shown).
Plp-CreERT2is a transgenic mouse line that uses
2.4 kb of the 5′ region, exon 1, and intron 1 of the
mouse proteolipid protein 1 gene to drive CreER
expression (Doerflinger et al. 2003). Several tamoxi-
fen induction paradigms have been characterized
with either the ROSA26LacZor ROSA26eYFPreporter
lines. Tamoxifen (33 mg/kg, IP) given once a day
from P0 to P7, P3 to P9, or P10 to P16 results in Cre+
inner phalangeal cells in the organ of Corti and Cre+
Schwann cells in the spiral lamina. The amount of
Cre+cells was not quantified (Gomez-Casati et al.
2010). We also characterized this line using the Ai14
reporter line (where deletion of the floxed “stop”
sequence drives expression of tdTomato; Madisen et
al. 2010). Tamoxifen (3 mg/40 g, IP) given once a day
at P0 and P1 results in 50 % tdTomato+inner
phalangeal cells in the apical turn and 80 % tdTo-
mato+inner phalangeal cells in the middle and basal
turns of the cochlea. We also found 5–10 % Tomato+
pillar and Deiters’ cells. No Cre+HCs were found
(Fig. 5A–C). We also found a large number of Cre+
Schwann cells in the spiral lamina (data not shown).
There was no effect on morphology or cellular
organization of the inner ear when BDNF was deleted
using the Plp-CreERT2allele and tamoxifen injections
were given once a day from P5 to P11 (Gomez-Casati
et al. 2010).
There are at least eight lines that use either the
mouse or human glial fibrillary acidic protein (GFAP)
promoter to drive the expression of Cre or CreER, of
which only one allele has been used in the inner ear.
The transgenic hGFAP-Cre line used 2.2 kb of the 5′
region from the human GFAP gene with the Gfa2
promoter to drive the expression of Cre (Zhuo et al.
2001). Using the mT/mG and ROSA26eYFPreporter
lines, Cre expression was detected in some SCs
postnatally, but the amount was not quantified (Hart-
man et al. 2010).
Cre/CreER LINES FOR THE POSTNATAL
VESTIBULAR SYSTEM
Several of the alleles that have Cre activity in the
postnatal cochlea are also expressed in vestibular
organs; however, less is known. This section will focus
on alleles where Cre activity occurs in the sensory
epithelia of vestibular organs after birth.
FIG. 4.
Representative confocal images of the organ of Corti from
Fgfr3-iCreERT2;Ai14 mice, induced with tamoxifen at P0 and P1. A
Slice image taken at the HC layer with tdTomato expression (red)
and HCs labeled by myosin VIIA (myo7a, green). B Slice image at
Fgfr3-iCreERT2expression pattern in the cochlea.
the SC nuclear layer with tdTomato expression (red) and SC nuclei
labeled by Hoechst (white). Scale bar, 10 μm. Quantification of
tdTomato+cells expressed as a percentage of total cells by type after
tamoxifen induction at P0/P1 (C), P2/P3 (D), P6/P7 (E), and P12/
P13 (F). DCs Deiters’ cells, PCs pillar cells, OHC outer hair cells.
COX ET.AL: Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP