C 2014 Wiley Periodicals, Inc.
V
genesis 52:695–701 (2014)
LETTER
Genomic DNA Recombination with Cell-Penetrating
Peptide-Tagged Cre Protein in Mouse Skeletal and
Cardiac Muscle
Wei-Ming Chien, Yonggang Liu, and Michael T. Chin*
Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington
Received 7 February 2014; Revised 1 April 2014; Accepted 15 April 2014
Summary: The Cre-loxP recombination system has
been used to promote DNA recombination both in vitro
and in vivo. For in vivo delivery, Cre expression is commonly achieved through the use of tissue/cell typespecific promoters, viral infection, or drug inducible
transcription and protein translocation to promote targeted DNA excision. The development of cell permeable (or penetrating) peptide tagged proteins has
facilitated the delivery of Cre recombinase protein into
cells in culture, organotypic slide culture, or in living
animals. In this report, we generated bacterially
expressed, his-tagged Cre protein with either a cardiac
targeting peptide or an antennapedia peptide at the Cterminus and demonstrated efficient uptake and
recombination in both cell culture and mice. To facilitate delivery to cardiac and skeletal muscle, we mixed
proteins with pluronic F-127 hydrogel and delivered Cre
protein into reporter Rosa26mTmG mouse skeletal
muscle or Rosa26LacZ cardiac muscle via ultrasound
guided injection. Activation of reporter gene expression
indicated that these Cre proteins were enzymatically
active. Recombination events were detected only in the
vicinity of injection areas. In conclusion, we have developed a method to deliver enzymatically active Cre protein locally to skeletal muscle and cardiac muscle that
may be adapted for use with other proteins. genesis
C 2014 Wiley Periodicals, Inc.
52:695–701, 2014. V
Key words: Cre recombinase protein; cardiac targeting
peptide; antennapedia peptide; pluronic F-127; cardiac
and skeletal muscle
The Cre-loxP system has been widely used for conditional gene targeting in mice (Nagy, 2000) and has led
to the development of many mouse lines used in the
study of cardiovascular development and disease models (reviewed in Doetschman and Azhar, 2012). Genera-
tion and characterization of tissue-specific and
inducible Cre transgenic lines is time consuming, and
can also be limited by unanticipated consequences.
Doxycycline used to induce expression, for example,
may attenuate cardiac hypertrophy in mouse models
(Errami et al., 2008) and overexpression of Cre driven
by the aMHC promoter can cause cardiac toxicity
(Agah et al., 1997; reviewed in Molkentin and Robbins,
2009).
The development of cell-penetrating peptides (CPPs)
linked to Cre has provided an alternative approach for
genomic manipulation. These peptides include HIV Tat
(YGRKKRRQRRR), antennapedia (RQIKIQFQNRRKWKK),
polyarginines (10 or 11 arginines), and FGF4 (AAVLLPVLLAAP) (Koren and Torchilin, 2012). Although Cre can
cross cell membranes without modification (Lin et al.,
2004; Will et al., 2002), tagging Cre with these CPPs
improves cell permeability. In addition, the positive
charges from the His-tag and nuclear localization signal
(NLS) of SV40 Tag also enhance the protein transduction
efficiency (Lin et al., 2004). With these improvements,
Cre-CPPs have been successfully used to promote recombination in mouse and human embryonic stem cell or
induced pluripotent stem cell lines (Haupt et al., 2007; Lin
et al., 2004; Nolden et al., 2006; Peitz et al., 2002), ex vivo
brain slide cultures (Gitton et al., 2009; Kim et al., 2009),
* Correspondence to: Michael T. Chin, Center for Cardiovascular Biology, University of Washington, School of Medicine, Box 358050, 850
Republican Street, Brotman 353, Seattle, WA 98109.
E-mail: [email protected]
Contract grant sponsor: Tietze Family Foundation; Contract grant
sponsor: John L. Locke Foundation
Published online 19 April 2014 in
Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/dvg.22782
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CHIEN ET AL.
FIG. 1. Expression and purification of His-Cre-CTP and His-CreANTP proteins. (a) Schematic diagram of CPP-tagged Cre proteins, which were constructed with 103 Histidine at the N-terminal,
a nuclear localization signal (NLS), and ANTP or CTP at the Cterminal with a linker peptide. (b, c) CPP-Cre proteins were
induced by IPTG and purified and buffer exchanged to 20% glycerol/PBS. His-Cre-ANTP (b) and His-Cre-CTP (c) proteins were
resolved in SDS-PAGE. Protein lysates from bacteria without
(Lanes 1 and 4) and with IPTG (Lanes 2 and 5) induction. Purified
and buffer exchanged His-Cre-ANTP (Lane 3) and His-Cre-CTP
(Lane 6).
and in vivo mouse models (Cronican et al., 2010; Jo et al.,
2001; Yu et al., 2003).
To explore the use of CPP-tagged Cre protein in skeletal and cardiac muscle, we designed two N-terminal
histidine tagged NLS-Cre CPP proteins (Fig. 1a), based
on recent findings (Lin et al., 2004; Peitz et al., 2002),
incorporating either an antennapedia peptide (ANTP)
or a cardiac targeting peptide (CTP, APWHLSSQYSRT).
The CTP was identified through bacterial phage display
screening and functions as a homing signal to cardiac
myocytes in vivo (Zahid et al., 2010). The bacterially
expressed His-Cre-CTP and His-Cre-ANTP proteins were
purified in native form and confirmed by SDS-PAGE
(Fig. 1b).
Cre protein recombination activity was assessed in
mouse embryonic fibroblasts (MEFs) isolated from
Rosa26mTmG/1 mice. These cells constitutively express
membrane-bound tdTomato fluorescent protein (mT),
but in the presence of Cre protein, undergo excision of
the open reading frame of mT, resulting in the expression of membrane-bound green fluorescent protein
(mG). The mTmG MEFs were incubated with His-CreCTP or His-Cre-ANTP or PBS control for 24 h and
assessed by fluorescence microscopy (Fig. 2a), flow
cytometry (Fig. 2b), and qPCR (Fig. 2c). About 13% of
MEFs were GFP positive at 130 mg/mL (2.9 mM) His-CreCTP. The percentage of GFP positive cells after His-CreCTP treatment was about four times that observed with
His-Cre-ANTP (Fig. 2b), suggesting that the type of CPP
tag affects Cre protein structure and recombination
activity. The kinetics of Cre CPP protein uptake was
also analyzed in Rosa26mTmG/1 MEFs. A 15-min incubation with either CPP-tagged Cre protein was sufficient
to promote a red to green fluorescent switch in some
cells (Fig. 3). Some double labeled cells were also
observed, which may be due to the prolonged protein
half-life of membrane-bound tdTomato (Muzumdar
et al., 2007). Our result showed that both tagged Cre
proteins can enter MEFs, however, the Cre-CTP protein
is more recombination efficient than Cre-ANTP.
To enhance delivery of protein to muscle tissue in
vivo, we mixed Cre protein in pluronic F-127 (PF-127,
also known as poloxamer 407), a thermosensitive
hydrogel. A 20% PF-127 hydrogel will keep in liquid
form at temperature below 24.6 C but will solidify at
body temperature. The amount of peptide that can be
released in vitro from such hydrogels ranges from 50 to
75% of material (Bromberg and Ron, 1998; Dumortier
et al., 2006).We injected 2.6 mg (20 mL in 20% PF-127/
13% glycerol/PBS) His-Cre-ANTP into the biceps femoris
of Rosa26mTmG/1 mice. Muscle was harvested 2 weeks
postinjection and sectioned for assessment of Cre activity detected by GFP fluorescence. Several GFP positive
myocytes were detected along the injection track (Fig.
4a). Next, we investigated whether PF-127 could facilitate uptake of protein into multiple muscles when
injected into the perimuscular space. His-Cre-ANTP was
injected through the tibialis anterior (TA) into the anterior tibial compartment. The control vehicle-injected
mice did not show any green fluorescent cells in the
extensor digitorum longus (EDL; Fig. 4b). A green fluorescent signal was observed at 2 weeks postprotein
injection in TA or EDL muscle (Fig. 4c), demonstrating
that His-Cre-ANTP can pass through epimysium and
perimysium, penetrate the cell membrane and promote
recombination. Similar results were observed with HisCre-CTP (Fig. 4d). Some myocytes were positive for
both fluorescent signals in the biceps femoris and EDL.
One possible explanation is that the recombination
event happened late, but this is unlikely because the
Cre-tagged protein can enter cells as rapidly as 15 min
in cell culture (Fig. 3) and PF-127 degrades within 2
days after subcutaneous injection (Hyun et al., 2007).
Other probable explanations are: (1) membrane bound
tdTomato in skeletal muscle has a long half-life as in
liver (9 days; Muzumdar et al., 2007); (2) Cre proteins
may require refolding once inside the cell and that may
require more time to become enzymatically active in
some cells; and (3) the recombination event happened
CRE-CPP MEDIATED RECOMBINATION IN VIVO
697
FIG. 2. In vitro Cre-CPP directed DNA recombination in Rosa26mTmG/1 MEFs. (a) MEFs were treated with PBS (left) or with 10 mg/mL HisCre-CTP (right). (b) MEFs were treated with 0.22 and 1.1 mM His-Cre-ANTP and His-Cre-CTP and 2.9 mM His-Cre-CTP. Percent green fluorescence positive cells obtained from duplicated experiments are shown. Y-axis: Average % EGFP-positive cells; X-axis: concentration of
Cre protein. (c) Quantitative PCR of genomic recombined mG allele relative to wild type Rosa26 allele and normalized to PBS-treated control. Primers were indicated as red arrows in (a). Scale bar: 250 mm. Yellow arrow indicates a double positive cell.
FIG. 3. Kinetics of Cre-CPP entry into MEF reporter cells. Rosa26mTmG/1 MEFs were incubated with His-Cre-ANTP (a) and His-Cre-CTP (b) for 15
and 30 min as indicated, washed and incubated with fresh media without Cre-CPPs for 24 h. Scale bar: 250 mm. Arrow indicates a double positive cell.
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CHIEN ET AL.
FIG. 4. Cre-CPP-mediated genomic DNA recombination in Rosa26mTmG/1 skeletal muscle. Mice received one injection of 20 mL 0.13 mg/
mL His-Cre-ANTP or 0.36 mg/mL His-Cre-CTP in 20% PF-127/13% glycerol/PBS in either the biceps femoris or through the TA into the
anterior tibial compartment. (a) Green fluorescent protein was visualized along the injection track in the biceps femoris muscle. (b) No
recombination in EDL was detected in vehicle-injected controls. (c) Recombination in EDL was observed after injection of 2.6 mg His-CreANTP, and (d) 7.2 mg His-Cre-CTP in the anterior tibial compartment. Scale bar: 250 mm. Arrows indicate double positive cells.
in at least one nucleus but not all in these multiply
nucleated skeletal myofibers.
To deliver protein to heart muscle, we chose
ultrasound-guided injection. We initially prepared Cre
protein with 20% PF-127 in a syringe kept on ice, then
placed the protein/syringe at room temperature for
3 min to promote a gel state. Once the protein/hydrogel
was injected, it started to solidify so that even when
minor leakage of the protein/hydrogel from the injection
site was detected, the leaked material formed a thin layer
on surface of the heart. With ultrasound guidance, we
injected approximately 18 mg His-Cre-CTP (50 mL of 0.36
mg/mL in 20% PF-127/13% glycerol/PBS or vehicle as
control) into the left ventricle wall of Rosa26LacZ/1 mice
(Fig. 5a–c). Mice were sacrificed at Day 4 postinjection,
to provide sufficient time for excision of the stop cassette in Rosa26LacZ/1 genomic DNA and expression of
LacZ (Soriano, 1999). Heart sections were stained with
X-gal to detect b-galactosidase activity. The LacZ positive
cells were not only detected around the injection track
but also spread out in surrounding cardiac myocytes
(Fig. 5d) probably because the Cre protein/hydrogel had
run through the interstitial space. Some lacZ positive
cells were detected along the epicardium, which likely
resulted from the solidified gel leaking around the injection site onto the surface of the heart (Fig. 5e).
We used relatively low amounts of Cre protein in cardiac (18 mg) and skeletal (2.6 mg) muscle injection (or
0.9 and 0.1 mg/kg, respectively, assuming the average
weight of a 12-week old mouse to be 25 g) in comparison with previous reports using intraperitoneal injection (25 mg/kg/day; Jo et al., 2001). The use of PF-127
hydrogel also limited the spread of injection fluid into
the chest cavity, which may compress the lung and lead
to death. Our local delivery methods using PF-127 can
easily be adapted to deliver other proteins to heart and
skeletal muscle. In conclusion, we have developed
novel Cre protein reagents that can enter muscle cells
and excise targeted alleles, and have further shown that
protein mixing with PF-127 hydrogel facilitates injection and uptake of enzymatically active protein into
skeletal muscle in living animals. When combined with
ultrasound guidance, the method can also be used to
deliver enzymatically active protein directly to cardiac
muscle. These reagents and delivery methods will allow
local gene targeting in cells and live mice and will also
facilitate local delivery of other proteins that may be
therapeutic for skeletal muscle and heart disorders.
METHODS
Plasmid Constructs
An N-terminal his-tag vector pET16b (Novagen, Billerica, MA) was used to make Cre expression constructs.
To generate pET16b-ANTP and pET16b-CTP vectors,
the 50 phosphorylated complementary oligomers
CRE-CPP MEDIATED RECOMBINATION IN VIVO
699
FIG. 5. His-Cre-CTP-PF-127 hydrogel mediated genomic DNA recombination in Rosa26LacZ mouse hearts. (a) Ultrasound-guided needle placement before (a) and after (b) injection of the mouse left ventricle wall. Yellow arrow: needle bevel; red arrow: injected protein mixture. No b-galactosidase activity was detected by X-gal staining in the vehicle-injected control (c) but was readily detected in the left
ventricle (d), and epicardium (e) after protein injection.
encoding a linker sequence and Drosophila melanogaster antennapedia motif ANTP (ESGGGGSRQIKIWFQNRRMKWKK; Zou et al., 2013) or CTP
(SGGGSPGAPWHLSSQYSRT; Zahid et al., 2010) were
synthesized and annealed to clone into pET16b (Novagen, Billerica, MA) at Xho I and BamHI sites. The open
reading frame of Cre recombinase with NLS was PCR
amplified from the genomic DNA of an aMHC-Cre transgenic mouse (Agah et al., 1997) with primers 50 -GTCATATGCCCAAGAAGAAGAGG-30
and
50 -AACTCGAG
0
ATCGCCATCTTCCAGCAGG-3 , with added Nde I and Xho
I restriction sites (underlined). The PCR product was
cloned into either pET16b-ANTP or pET16b-CTP to generate pET16b-Cre-ANTP (45.2 kd) or pET16b-Cre-CTP (44.6
kd). The constructs were confirmed by sequencing
(Operon). These reagents will be available to the scientific
community on acceptance of the manuscript.
M NaCl at 3 g wet weight/mL and stored at 280 C. Bacterial protein extracts were isolated with lysis buffer
(50 mM NaH2PO4, pH 8, 0.5 M NaCl, 10 mM imidazole,
0.2% Triton X-100, 6 mM 2-mercaptoethanol) at 1 g wet
weight per 5 mL buffer. The His-Cre-ANTP and His-CreCTP proteins were isolated with Ni-NTA superflow cartridges (Qiagen, Valencia, CA) or Hi-Pur cobalt resin
(Thermo Fisher, Waltham, MA). The nonspecifically
bound proteins were removed with wash buffer (50
mM NaH2PO4, pH 8, 0.3 M NaCl, 20 mM imidazole) and
the Cre proteins were eluted with elution buffer (250
mM imidazole in PBS). The eluted protein fractions
were buffer exchanged to PBS/20% glycerol with 10 K
Amicon centrifugal filters (Millipore, Billerica, MA) and
stored at 280 C. Protein concentration was determined
by 660 nm Protein Assay (Pierce, Rockford, IL).
Cre Recombinase Activity Assay
Protein Induction and Purification
Plasmids encoding His-Cre-ANTP and His-Cre-CTP
were transformed into BL21(DE3)pLysS (Invitrogen,
Carlsbad, CA) for protein expression. The overnight
bacterial cultures were inoculated at a dilution of 1:80 in
LB with 100 mg/mL ampicillin for 2 h at 37 C and proteins were induced with 0.5 mM IPTG for 2 h at room
temperature. The bacterial pellets were suspended in 5
MEFs were isolated from the dermis of embryonic
day 12.5 F1 C57BL6/129X1 Rosa26mTmG/1 embryos
(Muzumdar et al., 2007). For cell permeability kinetics,
MEFs were seeded in 24-well plates at 10,000 cells/well
with cover slides and incubated with 20 mg/mL (0.45
mM) His-Cre-CTP and His-Cre-ANTP for 15, 30, 60, 120,
or 480 min in 10% FBS/DMEM. More than two batch
purifications of each Cre protein were used. Cells were
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CHIEN ET AL.
then washed with media to remove Cre proteins and
cultured for an additional 24 h. Cells on cover slides
were mounted with Vectashield with DAPI (Vector Laboratories, Burlingame, CA). For recombination activity,
MEFs were seeded at 60,000 cells/well in 6-well plates
and incubated with 10, 50, and 130 mg/mL (or 0.22,
1.11, and 2.9 mM, respectively) Cre-CPP proteins. After
a 24-hours incubation, cells were harvested for flow
cytometry analysis. For genomic quantitative PCR on
the Rosa26mTmG/1 allele, MEFs were treated with 20
and 50 mg/mL for 24 h and DNA was isolated for qPCR.
The paired primers 50 -AAAGTCGCTCTGAGTTGTTAT-30
and 50 -GGAGCGGGAGAAATGATATG-30 were used for
detecting the unrecombined allele, whereas the primers 50 -GACACTAGTGAACCTCTTCGAG-30 and 50 AACAGCTCCTCGCCCTTGCTC-30 were used to detect
the recombined mG allele. The negative ddCt of the
recombined allele was calculated as relative to unrecombined allele and normalized to values from PBStreated MEF DNA. Data were collected from two
batches of purified proteins and duplicated wells of
MEF seeding 90,000 cells/well in 6-well plates.
In Vivo Cre-CPP Protein Injection in Skeletal and
Cardiac Muscle
A cohort of nine heterozygous F1 C57BL6/129X1
Rosa26mTmG/1 reporter mice at 12 weeks of age were
used for skeletal muscle injection. Mice were anesthetized with Avertin (0.5 g/kg). The procedure room was
maintained at 23 C. The purified His-Cre-ANTP was prepared in 20% PF-127/13% glycerol/PBS at a concentration of 0.13 mg/mL in a 0.3-mL hypodermic syringe
with a 29-gauge needle. Twenty microliter (2.6 mg) of
His-Cre-ANTP was injected directly into the biceps femoris muscle (three mice) or through TA into the anterior
tibial compartment (four mice). Similarly two mice
were injected with 20 mL 0.36 mg/mL His-Cre-CTP in
20% PF-127/13% glycerol/PBS through the TA into the
surrounding compartment. After 2 weeks, mice were
sacrificed and TA and EDL were isolated (Pasut et al.,
2013) and fixed in 2% paraformaldehyde/PBS for 10
min. Tissues were balanced stepwise with 10% sucrose/
PBS for >4 h and then 30% sucrose/PBS for overnight at
4 C and then embedded in OCT and frozen at 280 C.
A cohort of seven heterozygous inbred C57BL6
R0SA26LacZ/1 (Soriano, 1999) reporter mice at 12
weeks of age were used for in vivo cardiac intramuscular injection. The purified His-Cre-CTP was prepared in
20% PF-127/13% glycerol/PBS at a concentration of
0.36 mg/mL. Proteins were kept on ice in a 0.3-mL
hypodermic syringe with a 29-gauge needle to prevent
PF-127 solidification. The syringe was then warmed up
at room temperature for 3 min before ultrasoundguided cardiac intramuscular injection. Fifty microliter
(18 mg) of His-Cre-CTP was injected directly into the
left ventricle wall with direct echocardiographic
visualization (Vevo770, Visual Sonics, Toronto, Ontario,
Canada). On postinjection day 4, mice were sacrificed
and hearts were harvested, rinsed with PBS, embedded
in OCT, and frozen at 280 C.
X-Gal Staining and Immunofluorescence
Microscopy
To detect b-galactosidase activity, serial sections of
hearts from ROSA26LacZ mice were fixed with 2%
formaldehyde/0.2% glutaraldehyde and stained with
X-gal as described previously with a nuclear fast red
counterstain (Liu et al., 2012). To examine the red to
green fluorescent switch in ROSA26mTmG mice, serial
sections were viewed with either a Nikon Eclipse 80i
fluorescence microscope or with a Nikon TiE Inverted
Wildfield microscope.
ACKNOWLEDGMENT
The authors thank the Flow Cytometry Core facility at
UW South Lake Union, the Lynn and Mike Garvey Cell
Imaging Lab microscopy facility, and the Institute for
Stem Cells and Regenerative Medicine at the University of
Washington for technical support.
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