Shimshekgenesisi CRE2002
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TECHNOLOGY REPORT
Codon-Improved Cre Recombinase (iCre) Expression in
the Mouse
D.R. Shimshek,1 J. Kim,1 M.R. Hübner,2 D.J. Spergel,1 F. Buchholz,2 E. Casanova,3 A.F. Stewart,2
P.H. Seeburg,1 and R. Sprengel1*
1
Department of Molecular Neuroscience, Max-Planck Institute for Medical Research, Heidelberg, Germany
2
Gene Expression Program, EMBL, Heidelberg, Germany
3
Molecular Biology of the Cell I, German Cancer Research Center, Heidelberg, Germany
Received 24 September 2001; Accepted 20 November 2001
Summary: By applying the mammalian codon usage to karyotes, and it also contains an undesirably high fre-
Cre recombinase, we improved Cre expression, as de- quency of CpG dinucleotides— 65 CpG dinucleotides in
termined by immunoblot and functional analysis, in three the Cre gene—which can lead to epigenetic silencing
different mammalian cell lines. The improved Cre (iCre) during mammalian development (Cohen-Tannoudji et
gene was also designed to reduce the high CpG content
of the prokaryotic coding sequence, thereby reducing
al., 2000). Furthermore, expression levels are particu-
the chances of epigenetic silencing in mammals. Trans- larly important to applications of regulated recombi-
genic iCre expressing mice were obtained with good nases, either by regulated expression (Kuhn et al., 1995)
frequency, and in these mice loxP-mediated DNA re- or by regulated activity using fusion proteins of Cre with
combination was observed in all cells expressing iCre. ligand binding domains of steroid receptors (LBD; Logie
Moreover, iCre fused to two estrogen receptor hormone and Stewart, 1995). Therefore, we decided to optimize
binding domains for temporal control of Cre activity the codon usage of the Cre gene for use in mammals
could also be expressed in transgenic mice. However, (Haas et al., 1996). The altered version, termed iCre
Cre induction after administration of tamoxifen yielded (improved Cre), was assembled from oligonucleotides
only low Cre activity. Thus, whereas efficient activation
(Stemmer et al., 1995) to introduce silent base mutations
of Cre fusion proteins in the brain needs further improve-
ments, our studies indicate that iCre should facilitate corresponding to human codon-usage preferences, min-
genetic experiments in the mouse. genesis 32:19 –26, imize CpG content, eliminate putative cryptic splice
2002. © 2002 Wiley-Liss, Inc. sites (splice site prediction programs: www.cbs.dtu.dk/
services/NetGene2/ and www.fruitfly.org/seq_tools/
Key words: iCre recombinase; loxP; GnRH promoter; LBD; splice.html) and alter the stop codon. We also included
ER an optimal Kozak consensus sequence (Kozak, 1997),
and an additional valine codon in front of the simian
virus 40 large T nuclear localization signal (T-NLS, Fig. 1)
INTRODUCTION for potentially increased N-end rule stability (Varshavsky,
1997).
The bacteriophage P1-derived Cre recombinase (Cre) is In a first comparison, equal amounts of iCre and the
widely used to introduce specific gene deletions in se- prokaryotic Cre (Gu et al., 1994) expression vectors—
lected cell populations of genetically modified mice (re- both carried N-terminal the large T-NLS—were each tran-
viewed by Nagy, 2000). Despite a growing list of suc- siently transfected together with a lacZ expression plas-
cessful applications, recent experience indicates that mid in human embryonic kidney 293 (HEK293) cells.
several parameters can be critical for Cre-mediated re- From individual transfections the beta-galactosidase (-
combination efficiency at a given allele between two gal) activity was measured and showed transfection effi-
loxP-flanked target sites. These parameters include (i) ciency variations of about 0.5% (n ⫽ 9). When the
the distance between the two recombination target sites amount of Cre protein in the cell extracts was deter-
(Ringrose et al., 1999); (ii) genomic position effects that mined in immunoblots, normalized for -gal, the amount
may differ according to where the loxP sites are placed
in the genome and in which cell type recombination is
desired (Vooijs et al., 2001); and (iii) the level of Cre *Correspondence to: R. Sprengel, Department of Molecular Neuro-
expression in the target cell population. Here we aimed science, Max-Planck Institute for Medical Research, Jahnstrasse 29, 69120
Heidelberg, Germany.
to enhance the level of Cre recombinase expression in E-mail: sprengel@mpimf-heidelberg.mpg.de
mammalian systems. Because Cre is derived from a pro- Grant sponsors: Deutsche Forschungsgemeinschaft and the Volkswagen
karyotic source, its codon usage is not optimal for eu- Foundation.
20 SHIMSHEK ET AL.
of iCre was 1.58 ⫾ 0.15 (mean ⫾ SEM; P ⬍ 0.05; n ⫽ 6) Cre-LBD fusion proteins before activation by ligands (An-
fold higher than that of Cre expressed from the same grand et al., unpublished results). After selection for
vector (Fig. 2a). Increased iCre expression was con- stable integration of the Cre-ABD plasmids, pools of
firmed in an enzymatic test (Fig. 2b). Protein extracts of primary colonies were taken for immunoblot analysis
transfected cells were incubated with a substrate for Cre (Fig. 3b). In two experiments, iCre-ABD expression lev-
in the form of linearized DNA containing two loxP sites els were 2.5 ⫾ 0.5 (P ⬍ 0.05) greater than Cre-ABD
and releasing a 2,895 bp fragment upon Cre recombina- expression levels. The ES cells used for this experiment
tion. In agreement with the enhanced expression levels were of the Ex3 line that carries a stably integrated
of iCre (Fig. 2a), DNA recombination was 1.83 ⫾ 0.18 loxP/lacZ recombination reporter in which -gal reports
fold higher (P ⬍ 0.01; n ⫽ 2; -gal normalized) by recombination (Kellendonk et al., 1996). Immediately
extracts containing iCre than Cre (Fig. 2b). Transfection after electroporation, 10⫺7 M mibolerone was added to
experiments with CV1/lacZ indicator cells (Ludwig et plates so that iCre-ABD or Cre-ABD would be induced
al., 1994) yielded a similar result (Fig. 2c). In this assay, while stable, G418 resistant colonies arose. The plates
Cre activity removed the floxed stop insert and activated were then stained for -gal expression in situ. Of 158
-gal expression. Again, iCre had 1.68 ⫾ 0.02 (P ⬍ 0.01; G418-resistant colonies on the Cre-ABD plate, 52 (32.9%)
n ⫽ 2) fold higher activity than Cre (Fig. 2c). In this showed -gal expression, whereas on the iCre-ABD a
experiment an expression plasmid coding for alkaline 1.8-fold increase of colonies (146/155, 94.2%) were pos-
phosphatase (AP) was cotransfected. The number of itive for -gal despite its high basal activity (basal activity
AP-positive cells was used to normalize for transfection without inducing ligand was 0.2% for Cre-ABD and
efficiency. 34.6% for iCre-ABD). This result supports the immuno-
To establish whether the improved expression prop- blot analysis and indicates that iCre can improve expres-
erties of iCre could confer improved expression onto a sion levels of Cre-LBD but increase the basal activity of
Cre-LBD fusion protein, and also to test expression from the ABD fusion protein.
stably integrated transgenes, plasmids encoding Cre-ABD Based upon the improved properties of iCre in cell
(androgen receptor binding domain) or iCre-ABD were culture experiments, we verified functionality in trans-
constructed (Fig. 3a) and stably integrated into mouse genic mice and expressed iCre via a mouse gonado-
embryonic stem (ES) cells. The N-terminal NLS was re- tropin-releasing hormone (GnRH) promoter fragment. In
moved from Cre and iCre for this experiment because its previous studies using this fragment (Spergel et al.,
presence increases background Cre recombination by 2001) or similar ones (Pape et al., 1999), GnRH pro-
22 SHIMSHEK ET AL.
FIG. 3. Analysis of stably expressed iCre in mouse ES cells. (a) Diagram of the linearized plasmid pBKC-iCre-ABD (6,711 bp) used for stable
integration. hCMV (late enhancer/promoter cassette of human CMV); neo (kanamycin/neomycin resistence gene). (b) Immunoblot analysis
(two experiments) from total cell extracts of Ex3 ES cells. The two experiments differed in the presence (Exp 1) or absence (Exp 2) of 1 g/ml
puromycin co-selection with G418. (c) The sequence of the 5⬘-end of the iCre-ABD and Cre-ABD coding regions. Both iCre-ABD and
Cre-ABD genes carry this sequence at their 5⬘-ends. Translational start codon is encircled.
moter activity in the mouse brain was observed in most Indra et al., 1999) via the GnRH promoter to achieve
GnRH immunopositive neurons and in a second popula- temporal regulation of iCre activity. Again we ob-
tion of cells about 18 times larger than the GnRH ex- tained high (n ⫽ 3) and low (n ⫽ 2) expressing lines
pressing population. When we used this GnRH promoter (TgGnRH-ERiCreER). In all lines, tamoxifen induced nuclear
fragment to drive nuclear iCre expression, we obtained accumulation of the Cre immunostain (Fig. 5b, c, d),
four mouse lines exhibiting high expression, three with indicating that the estrogen receptor antagonist released
low expression, and two with no iCre expression. In all the iCre fusion protein from its hsp70-mediated cytoplas-
four high-expressing lines, cell nuclei of GnRH neurons mic localization. In tamoxifen-injected offspring of
and neurons of the second population were immunopo- TgGnRH-ERiCreER mice containing the lacZ gene of
sitive for iCre (Fig. 4c, e, f). Double labeling with GnRH GTRosa26 mice we detected loxP-directed recombina-
and Cre antisera confirmed cellular colocalization of cy- tion at the lacZ locus of the indicator gene by PCR of
toplasmic GnRH and nuclear Cre in 89 ⫾ 5% (n ⫽ 5 total brain DNA (data not shown). However, -gal ex-
mice) of GnRH neurons (Fig. 4e). pression in GTRosa26/TgGnRH-ERiCreER mice could not be
Cre-activated -gal expression as determined by X- detected indicating that the activity of iCre recombinase
Gal staining was observed in offspring of transgenic is too low. Hormone-binding domains flanking Cre re-
(TgGnRH-iCre) mice bred with a Cre indicator mouse duce the recombination efficiency in vitro (Kellendonk
line (GTRosa26; Soriano, 1999). In double positive et al., 1996), and it may be further reduced in a living
(GTRosa26/TgGnRH-iCre) mice, Cre activity was detected organism. In addition, the chromatin structure may
in 97 ⫾ 0.5% (n ⫽ 2 mice) of GnRH neurons and also in hinder the accessibility of the loxP elements in the
the second population (Fig. 4d, g). Thus, in the first test mouse genome, thereby reducing the effectiveness of
in mice, iCre presented good operational properties. ERiCreER to act on these loxP sites. As a result, recom-
Neurons in other areas, mainly of the limbic system, also bination events at the Cre indicator gene in GTRosa26/
exhibited Cre activity. This widespread iCre activity seen TgGnRH-ERiCreER may occur in only a few cells not detect-
in GTRosa26/TgGnRH-iCre mice may reflect in part a de- able by X-Gal staining.
velopmental regulation of the GnRH promoter (Skynner In summary, in our mammalian cell culture studies,
et al., 1999). In the adult GTRosa26/TgGnRH-iCre mouse, iCre was expressed better than prokaryotic Cre and also
Cre protein is only detected in GnRH neurons and in better than the humanized Cre described by Koresawa et
neurons of the second population. Thus, later in devel- al. (2000). In our transgenic mouse model TgGnRH-iCre
opment, the activity of the GnRH promoter might be iCre was expressed in four out of nine founders at high
more restricted. levels and with high activity. Thus, adapting codon usage
Consequently, we expressed iCre flanked by two es- elevated Cre expression, as was the case with GFP from
trogen receptor-binding domains (ER; Zhang et al., 1996; jellyfish (Zolotukhin et al., 1996) and with the lac repres-
CODON-IMPROVED CRE 23
FIG. 4. GnRH–iCre construct and its expression in transgenic mice. (a) GnRH-iCre construct (5,028 bp) used to generate TgGnRH-iCre mice.
(b) Schematic diagram of a mouse brain slice. cc, corpus callosum; CPu, caudate putamen; MS, medial septal nucleus; LS, lateral septal
nucleus; ac, anterior commissure; POA, preoptic area. (c) Expression and colocalization of iCre and GnRH in a coronal section. oc, optic
chiasm. (d) Immunopositive GnRH neurons and -gal expressing cells in GTRosa26/TgGnRH-iCre mice. (e, f, g) Higher magnification showing
GnRH- (blue) and iCre-positive (brown) cells in the POA (e), iCre-positive cells in the LS (f), -gal and GnRH expression in the POA (g).
sor (Cronin et al., 2001). We conclude that iCre, when sembled by PCR (Stemmer et al., 1995). Fragments of the
expressed in select tissues and cell populations expected length (1.091 kb) were isolated, reamplified,
(Casanova et al., 2001), is well suited for studying gene and sequenced with primers C0 (5⬘-GAGGAAGCTTGTC-
function in the mouse. CACCATGGTGC-3⬘) and R0 (5⬘-CGCTCCGTCGACT-
CAGTTTCAGTC-3⬘).
MATERIALS AND METHODS
Plasmids
Synthesis of the Improved Cre The iCre gene was cloned into a prokaryotic expres-
To synthesize the iCre gene (Fig. 1, Genbank acces- sion vector composed of pBAD (Invitrogen, Groningen,
sion no. AY056050), the Cre DNA sequence of pMC-Cre The Netherlands) and part of pPKM-6 (Reiss et al., 1984)
(Gu et al., 1994) was modified. Twenty-four 70-mer via HindIII and SalI and introduced into DH5␣ cells
oligonucleotides encoding the iCre sequence were as- containing the Cre indicator plasmid pSVpaX1 (Buchholz
24 SHIMSHEK ET AL.
FIG. 5. GnRH-ERiCreER construct and its expression in transgenic mice. (a) GnRH-ERiCreER construct (7,350 bp) used to generate
TgGnRH-ERiCreER mice. (b) Expression and colocalization of iCre and GnRH in a coronal section of tamoxifen-treated TgGnRH-ERiCreER mice.
LS, lateral septal nucleus; MS, medial septal nucleus; DBB, diagonal band of Broca; ac, anterior commissure. (c) Higher magnification
showing cells in the DBB that are iCre (brown) and GnRH (blue) immunopositive after tamoxifen treatment (inset in (c): iCre in the DBB
without tamoxifen), (d) cells in the LS that are iCre immunopositive after tamoxifen treatment (inset in (d): iCre in the LS without tamoxifen).
et al., 1996). Out of 31 recombinant clones, two harbored activity of 1 g Cre expression plasmid is given as the
the expected sequence. Plasmid pL29mGnRH.iCre was number of blue cells/number of AP-positive cells. Ex3 ES
constructed by substituting the GFP fragment in the cells (5 ⫻ 106) were electroporated (240V, 500 F) with
plasmid pL29mGnRH.hGFP2 (Spergel et al., 1999) by 20 g Cre-ABD or iCre-ABD plasmids, linearized by
iCre via HindIII and SalI. Plasmid pL29mGnRH.ERiCreER ApaL1. Selection (G418, 200 g/ml) was initiated 1 day
was constructed as described above except that the after plating.
GFP-SV40 sequence was substituted by the ERiCreER-
hghpolyA (polyadenylation signal of the human growth Protein Preparation and Immunoblot Assays
hormone) sequence via a 5⬘-blunt and 3⬘-BssHII ligation. Protein from transfected HEK293 cells was prepared
Plasmid pBKC-iCre-ABD was constructed by substituting 24 h after transfection and immunoblots (10% separating
Cre with the 5⬘-altered iCre fragment via HindIII and and 4% stacking SDS-polyacrylamide gels) were per-
XhoI in pBKC-Cre-ABD. formed. For immunoblotting of Ex3 ES cells, Ex3 cells
were harvested by trypsinization. 2 ⫻ 106 cells were
Transfection and Staining Assays pelleted; resuspended in 1 ml 250 mM Tris-HCl, pH 7.5;
HEK293 cells were transfected with plasmid pRK supplemented with 1mM ethylenediaminetetraacetic
(Schall et al., 1990) containing the iCre or Cre recombi- acid (EDTA), 1 mM phenylmethanesulfonyl fluoride
nase gene (5 g) and cotransfected with pCMV-lacZ (5 (PMSF), and 1 mM dithiothreitol (DTT); and frozen in a
g) to monitor transfection efficiency by -gal (Miller et dry ice/ethanol bath, thawed in a 37°C bath twice. NaCl
al., 1992). The pHD vectors (Kellendonk et al., 1996) was added to 400 mM, freeze/thaw cycle repeated twice
encoding iCre and Cre (2 g) were transfected into CV1 and centifugated at 1,600⫻g for 4 min (4°C). Protein in
cells carrying a lacZ indicator plasmid (Ludwig et al., supernatant was precipitated with 10% ice-cold tri-
1994) and cotransfected with pHD-AP (1 g) for con- chloracetic acid (TCA) overnight (4°C) and centrifugated
trolling transfection efficiency by Fast Red staining at 16,000⫻g for 10 min (4°C). Precipitate was washed
(Roche, Mannheim, Germany). -gal activity was de- twice in ice-cold acetone, dried and separated by 10%
tected with X-Gal solution (Kellendonk et al., 1996). Cre SDS-polyacrylamide (5% stacking zone) for immunoblot-
CODON-IMPROVED CRE 25
ing. Quantification was performed with Image Reader ACKNOWLEDGMENTS
BAS1000 (Fuji) and Image Gauge 3.0.
We thank A. Herold for making the GnRH-iCre construct,
In vitro Recombination Assay Dr. R. Benoit for GnRH antibody LR-1, F. Zimmermann
for pronucleus injections, Dr. P. Soriano for lacZ re-
Equal amounts of protein extracts from each transfec- porter mice, Drs. Y. Gu and K. Rajewsky for Cre cDNA,
tion were incubated with equal amounts of AflIII-linear- and Dr. A. Nagy for pLoxPNeo-1. D. R. S. was a recipient
ized 4,995 bp plasmid pLoxPNeo-1 (Nagy et al., 1998) as of a Deutsche Forschungsgemeinschaft (DFG) long-term
described by Lee and Saito (1998). Recombination fellowship. D. J. S. was supported by a SFB grant from
events were analyzed on a 1% agarose gel, scanned, the DFG. Funded, in part, by a grant from the Volkswa-
quantified with Image Gauge 3.0, and normalized for gen Foundation to R. S. and P. H. S. Experiments were in
transfection efficiency. accordance with protocol 35-9185.81/35/97 approved
by the Regierungspraesidium Karlsruhe, Germany.
Generation of Transgenic Mice
LITERATURE CITED
Transgenic founders TgGnRH-iCre and TgGnRH-ERiCreER
were generated by pronucleus injection of linearized
Buchholz F, Ringrose L, Angrand PO, Rossi F, Stewart AF. 1996.
(AvrII/NsiI) and purified pL29mGnRH.humCre and Different thermostabilities of FLP and Cre recombinases: implica-
pL29mGnRH.ERiCreER minigenes (Spergel et al., 1999). tions for applied site-specific recombination. Nucleic Acids Res
TgGnRH-iCre and TgGnRH-ERiCreER mice were selected by 24:4256 – 4262.
PCR analysis of tail DNA (Spergel et al., 1999) with Casanova E, Fehsenfeld S, Mantamadiotis T, Lemberger T, Greiner E,
Stewart AF, Schutz G. 2001. A CamKIIalpha iCre BAC allows
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GAAG-3⬘) and iCre32 (5⬘-CACAGACAGGAGCATCTTC- Cohen-Tannoudji M, Vandormael-Pournin S, Drezen J, Mercier P, Babi-
CAG-3⬘), which amplified a 414 bp DNA fragment or net C, Morello D. 2000. lacZ sequences prevent regulated expres-
with EiCre1 (5⬘-GACAGGCAGGCCTTCTCTGAA-3⬘) and sion of housekeeping genes. Mech Dev 90:29 –39.
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