© 2000 Wiley-Liss, Inc.
genesis 28:93–98 (2000)
TECHNOLOGY REPORT
Cre Recombinase Expression in Cerebellar Purkinje Cells
Jaroslaw J. Barski,1,2* Kathrin Dethleffsen,1 and Michael Meyer1
1
Max-Planck-Institute of Neurobiology, Martinsried, Germany
Medical University of Silesia, Department of Physiology, Katowice, Poland
2
Received 5 September 2000; Accepted 16 September 2000
Summary: The cerebellar cortex and its sole output, the
Purkinje cell, have been implicated in motor coordination, learning and cognitive functions. Therefore, the
ability to generate Purkinje cell-specific mutations in
physiologically relevant genes is of particular neurobiological interest. A suitable approach is the Cre/loxP
strategy that allows temporally and spatially controlled
gene inactivation. Here, we present the characterization
of transgenic mouse strains expressing Cre recombinase controlled by the L7/pcp-2 gene. Endogenous L7/
pcp-2 protein is expressed exclusively in Purkinje cells
and retinal bipolar neurones. Recombination was detected by ␤-galactosidase histochemistry in tissues
from crosses of the L7/pcp-2:Cre transgenic lines with
two different indicator strains, GtROSA26 and ACZL.
Purkinje cells in all folia of the cerebellum displayed
intense ␤-galactosidase staining, whereas only few blue
cells were observed in the retina and other parts of the
CNS. Thus, these transgenic lines are potentially of great
importance for genetic manipulations in cerebellar Purkinje cells. genesis 28:93–98, 2000.
© 2000 Wiley-Liss, Inc.
Key words: Cre recombinase; transgenic mouse; cerebellum; Purkinje cells; pcp-2; L7
Conditional mutagenesis via the Cre/loxP or Flp/Frt system critically depends on the spatial and temporal expression profile of the recombinases Cre or Flp. Conditional gene targeting in the central nervous system of
postnatal mice is a particular challenge as the questions
addressed often aim at physiologically and functionally
defined, mostly small subpopulations of neurons, for
which single specific genetic markers are not known
(and are even unlikely to exist). A general solution to this
dilemma may be the development of approaches where
Cre activity depends on coexpression of several transgenes, each driven by different regulatory elements.
These are chosen such that their activities overlap only
in the neuronal subpopulation of interest, thus constituting a combinatorial code. As these approaches have
yet to be developed, there are few examples of neuronal
subpopulation-specific Cre expression in postnatal mice.
Cerebellar Purkinje cells are the principal neurons of
the cerebellar cortex and its exclusive efferents . They
are thought to be involved in motor coordination and
motor learning but they may also participate in cognitive
functions ( in Houk, 1997; Mauk, 1997). These neurons
are an exception to the problem described above, as
there are several genes that are predominantly expressed
in Purkinje cells. These include the glutamate receptor
delta 2 (Araki et al., 1993; Lomeli et al., 1993) and the
L7/pcp-2 gene (Nordquist et al., 1988; Oberdick et al.,
1988). Here, we describe the generation and characteristics of L7/pcp-2: Cre transgenic mice. L7/pcp-2 is a
protein of unknown function expressed exclusively and
abundantly in postnatal Purkinje cells and retinal bipolar
neurones of several species (Nordquist et al., 1988; Oberdick et al., 1988; Berrebi et al., 1991). Recent biochemical analysis suggested a role of this protein as a
guanine nucleotide exchange factor for G␣o and related
G proteins (Luo et al., 1999). Nullmutants generated by
two independent groups failed to reveal any abnormalities (Mohn et al., 1997; Vassileva et al., 1997). However,
as we could not exclude yet unknown phenotypes associated with loss of this gene, we preferred a transgenic
approach using a previously described minigene
(Fig.1A)(Smeyne et al., 1995) rather than a knock-in that
may affect expression of this gene and thereby possibly
disrupt cerebellar functions. Previous experiments have
shown that L7 gene constructs can direct expression of
heterologous genes to Purkinje cells of postnatal mice
(Smeyne et al., 1991, 1995; Buffo et al., 1997; De Zeeuw
et al., 1998; Baader et al., 1999). It was, however, unclear whether temporal and spatial specificity would
allow use of this minigene to induce Cre-mediated recombination specifically in postnatal Purkinje cells. The
Cre cDNA was inserted into exon 4 of the L7 minigene,
the construct linearized and used to electroporate R1
embryonic stem cells (Nagy et al., 1993). We have chosen a transgenic approach via embryonic stem cells as
we wanted to avoid complex, potentially instable multicopy transgene arrays that are often observed after pronucleus injection. Three clones (L7Cre-2, L7Cre-8,
L7Cre-12) were selected for blastocyst injection and con*Correspondence to: Jaroslaw J. Barski, Max-Planck-Institute of Neurobiology, Dept. of Neurobiochemistry, Am Klopferspitz 18A, D-82152 Martinsried, Germany.
E-mail: [email protected]
Contract grant sponsors: Max-Planck-Society and Deutsche Forschungsgemeinschaft; Contract grant numbers: ME1121/3-1 and ME1121/3-2.
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FIG. 1 Transgene construct. (a) Insertion of Cre-recombinase cDNA into the L7⌬AUG-vector. Black boxes indicate the four exons and the
arrowhead to the left of exon 1 the transcription start of the L7 gene. Waved lines are plasmid sequences. (b) Southern blot analysis of the
L7Cre-2 transgenic line.
tributed to the germline of the resulting chimeras. Southern blotting of F1 offspring revealed integration of few
transgene copies in all lines (Fig.1B, data not shown).
Offspring of all lines was fertile and transgene transmission followed Mendelian rules. Immunostaining with a
polyclonal Cre antibody (Kellendonk et al., 1999) demonstrated the exclusive presence of the protein product
in cerebellar Purkinje cells in all three lines (Fig. 3A, F).
For further investigation, line L7Cre-2 was selected. CremRNA was readily detectable in nonradioactive Northern blots of cerebellar samples of transgenic but not
wild-type mice (not shown). Specificity of Cre expression for Purkinje cells was verified by nonradioactive in
situ hybridization using Cre-specific riboprobes (not
shown). Because we wanted to use offspring from crossings of these mice with floxed lines also in behavioral
paradigms, homozygous L7Cre-2 mice and their wildtype littermates were subjected to two behavioral tests.
In the open field, which provides information about
general locomotor abilities as well as exploration and
anxiety-related behaviors, homozygous mutants could
not be distinguished from their wild-type littermates
(Fig. 4A, B). The same was true for the runway assay
where mice have to transverse a 1-m long and 2-cm wide
bar the surface of which is provided with evenly spaced
0.5-cm high obstacles (see Kashiwabushi et al., 1995;
Airaksinen et al., 1997). With this test, dysmetria/ataxia
related behaviours can be evaluated (Fig. 4C). To check
for morphological integrity of Cre-expressing Purkinje
cells we have performed immunohistochemistry for a set
of proteins normally expressed in different cellular compartments of Purkinje cells. Staining for calbindin and
PEP19, two preferentially cytosolic calcium-binding proteins (Ziai et al., 1988; Baimbridge et al., 1992), for
PKC-␥, which when activated is membrane associated
(Kose et al., 1988), and GAD, the key enzyme required
for synthesis of the Purkinje cell’s neurotransmitter
GABA (Chan-Palay et al., 1983), failed to reveal any
differences in distribution and intensity between heterozygous mutants and their wild-type littermates (Fig.
3B-E, G-J).
To further test the specificity of Cre-mediated expression, L7Cre-2 transgenic mice were mated with two
strains of indicator mice: the GtROSA26 or ACZL- indicator strain (Akagi et al., 1997; Soriano, 1999). To determine the localization of recombination events, various
tissues of adult L7Cre-2/⫹; GtROSA26/⫹, and
L7Cre-2/⫹; ACZL/⫹ mice were analyzed for expression
of ␤-galactosidase by X-gal staining. Mutants lacking the
L7Cre-2 allele, i.e., GtROSA26/⫹ and ACZL/⫹, served as
controls. Tissues of both control strains were devoid of
blue cells. In the cerebellum of L7Cre-2/⫹;GtROSA26/⫹
animals, Purkinje cells in all folia displayed very intensive
X-gal-positive reaction. The whole soma of these neurons was filled with the reaction product (Fig. 2A, B, C).
Dispersed granule-like blue structures were also found in
the molecular layer of the cerebellar cortex where phase
contrast illumination demonstrated their association
with the dendritic arborizations of Purkinje cells (Fig.
2D). This localization is presumably caused by transport
of the reaction product along the dendritic processes as
has also been observed with other lacZ insertions (e.g.,
Fässler and Meyer, 1995). ␤-galactosidase positive cells
were rarely observed in the granular layer of the cerebellar cortex, deep cerebellar nuclei and inferior olive
(Fig. 2D, H, G). No attempt has been made to identify the
labeled cells in these structures that may represent glial
or neuronal cell bodies or, in case of the granular layer,
Purkinje cell axons. A low number (⬍ 5%) of X-galpositive cells was visible in the cerebral cortex, dentate
gyrus, pyramidal layers of the hippocampus, and in the
striatum (Fig. 2E, I, J, F). In all these areas label appeared
to be randomly distributed without any spatial preferences. Because L7 expression in bipolar neurones of
mouse retina is well established (Berrebi et al., 1991),
we looked for recombination events in that structure.
Surprisingly, only few ␤-galactosidase-positive cells
could be detected in the inner nuclear layer (Fig. 2K).
Recombination was also present in some non-neuronal
organs, such as the kidney, where labeling was evident
in a low number of tubular structures (Fig. 2L). Developmentally, X-gal positive cells were observed for the
first time in the Purkinje cell layer at postnatal day 6 (Fig.
2M). Analysis of double heterozygous animals from
crossings of the L7Cre-2 with the ACZL indicator line
confirmed labeling of Purkinje cells although staining
appeared to be lower in intensity and more granular than
with the other indicator mouse (data not shown). It was
restricted to Purkinje cell somata. Analysis of all the
other brain regions failed to reveal any blue cells in this
cross. Thus, it appears that the ACZL transgene, which is
controlled by the chicken actin promotor, cannot be
activated in all brain regions after removal of the floxed
stop cassette.
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FIG. 2 X-gal staining of adult (a)–(l) and early postnatal (m) L7Cre-2/⫹;GtROSA26/⫹ mice. (a) Midsagittal section of the cerebellum, bar ⫽
5 mm. (b)–(d) Higher magnification of the framed areas. (b) bar ⫽ 100 ␮m. (c) bar ⫽ 50 ␮m. (d) bar ⫽ 25 ␮m, staining product granules in
the molecular layer of the cerebellar cortex are indicated by arrowheads. (e)–(l) ␤-galactosidase-positive cells (arrowheads) in: (e) parietal
cortex, (f) striatum, (g) inferior olive, (h) deep cerebellar nuclei, (i) dentate gyrus, (j) CA1 area of the hippocampus, (k) retina, (l) kidney, (m)
stained Purkinje cells at postnatal day 6. (e–j, l) bar ⫽ 50 ␮m, (k) bar ⫽ 25 ␮m, (m) bar ⫽ 10 ␮m.
In summary, we have generated a mouse strain that
efficiently expresses Cre recombinase in almost all cerebellar Purkinje cells. Cre-mediated recombination was
first observed around postnatal day 6 and fully established by 2 to 3 weeks after birth. The potential impact
of the low number of ␤-galactosidase expressing cells
which were not Purkinje cells has to be evaluated for
each floxed allele as their number may be too low to be
relevant for the biological question addressed or they
may not at all express the floxed gene.
additional two generations. ACZL mice on an FVB background were obtained from the European Mouse Mutant
Archive (Monterotondo, Italy) via its node CDTA (Orleans, France) and have been backcrossed to C57Bl/6 in
the laboratory for one or two generations. C57Bl/6 mice
were purchased from Charles River (Sulzbach, Germany), strain designation: C57Bl/6NCrl BR.
METHODS
Plasmids
The L7⌬AUG plasmid was obtained from John Oberdick (Smeyne et al., 1995) and the pMC-Cre plasmid
from H. Gu (Gu et al., 1993).
Mouse Lines
The GtROSA26 line was obtained from the Jackson
Laboratories (Bar Harbor, ME) (stock designation: B6,
129S-Gtrosa26具tm1Sor典; stock # 003309) and has been
backcrossed to C57Bl/6 mice in the laboratory for an
Antibodies
The PEP-19 antibody was obtained from Karl Heinz
Herzog and Jim Morgan (Ziai et al., 1988) and the Creantibody from Christian Kellendonk and Günther Schütz
(Kellendonk et al., 1999).
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FIG. 3 Immunodetection of Cre protein and Purkinje cell markers in the cerebellar cortex of adult mice: (a, f) Phase contrast microphotographs of Cre-recombinase staining in the nuclei of Purkinje cells (arrowheads), bar ⫽ 15 ␮m. Confocal images of immunoreactivity for
(b, g) calbindin D28k, (c, h) PEP-19, (d, i) PKC-␥, (e, j) GAD, bars ⫽ 20 ␮m. Genotypes are indicated in the upper left corner.
Generation of the Transgenic Mouse Lines
To simplify further cloning steps, the EcoR I / Hind III
fragment of the vector L7⌬AUG, were subcloned into a
Bluescript SK vector lacking the BamH I restriction site.
To obtain the L7Cre transgene, the Cre cDNA of pMCCre was inserted into the BamH I site of the fourth exon
of the L7 minigene (Fig. 1A). To enable selection, R1
embryonic stem cells (obtained from A. Nagy) were
coelectroporated with the EcoR I/Hind III fragment of
L7Cre and a neomycin resistance plasmid. G418-resistant
colonies of embryonic stem cells were screened for the
presence of transgenic DNA by Southern blot hybridization using a Cre-specific probe, and positive clones were
injected into C57Bl/6 blastocysts using standard procedures (Hogan et al., 1994). The resulting chimeras were
crossed with C57Bl/6 mice to obtain the F1-generation.
Four of eight clones contributed to the germ line and
three of these (L7Cre-2, L7Cre-8, L7-Cre-12) were used to
establish mutant lines.
Genotyping of the L7Cre transgenic mouse lines was
made by Southern blotting. DNA from tail biopsies were
digested with Hinc II and hybridized with a probe specific for the L7 minigene (Fig. 1A, B).
infrared sources and sensors (beam spacing 1.52 cm)
mounted to register all horizontal and vertical (rearing)
movements. For each mouse, the whole trial lasts 30 min
subdivided into 1-min bins. Distance parameters presented in Fig. 4A are defined as the sum of distances
between successive coordinates in the X-Y (horizontal)
plane. Values on the Y-axis of the plot are the mean
distances (n ⫽ 6 mice for each group) travelled in each
single 1-min bin. The X-axis gives the time in minutes.
The second parameter analysed was the vertical activity of the animals, recorded during the same experimental trials. This kind of movement is depicted in Figure 4B
as “vertical breaks,” which were defined as the total
number of disruptions of infrared beams of the upper
sensor ring, which can only occur if the mouse stands on
its hind extremities having no ground contact with the
forelimbs.
The runway test was performed as described (Kashiwabuchi et al., 1995; Airaksinen et al., 1997). For each
mouse the test was performed on five consecutive days.
Each day, each mouse (n ⫽ 6) underwent five consecutive trials. Slips were counted on one side.
All obtained data were statistically analyzed with the
ANOVA-single factor test.
Behavioral Analysis
Open field analysis was performed by means of the
multiparameter activity monitor Tru Scan and its associated software (Coulbourn Instruments, Allentown, PA).
To record the activity, the mouse is placed on a cagearena (25.9 ⫻ 25.9 cm) equipped with two rings of
Histochemistry
The X-gal staining was performed according to standard procedures (Michaelidis et al., 1996). To enable
better localization of positive signals, tissue slices were
subsequently counterstained with neutral red.
RECOMBINATION IN PURKINJE CELLS
97
FIG. 4 Behavioral analysis of adult homozygous transgenic mice. (a) Horizontal motor activity, during the 30-min open field test session.
The values are average distances for n ⫽ 6 mice in each group. (b) Vertical activity as the total number of vertical breaks during 30 min of
the open field test. The values are average numbers of breaks for n ⫽ 6 mice for each group. (c) Runway task. Average number of slips over
the 5 consecutive days of the test, n ⫽ 6 mice for each group.
Immunohistochemistry
In the present study the following antibodies were
used: rabbit anti Cre-recombinase 1:5,000 (Kellendonk
et al., 1999); rabbit anti GAD 1:200 (Chemicon, Temecula, FL); rabbit anti PEP-19 1:400 (Ziai et al., 1988);
rabbit anti PKC-␥ (SIGMA, Deisenhofen, Germany) and
mouse anticalbindin Cb28k 1:500 (SWant, Bellinzona,
Switzerland).
Immunostaining for Cre-recombinase were made
following a previous protocol (Kellendonk et al.,
1999). Immunostaining for GAD, PEP-19, PKC-␥, and
calbindin were made on a 30␮m thick free-floating
section according to standard protocols (Arai et al.,
1993). Immunoreactivity was visualized with anti-rabbit or anti-mouse secondary antibodies coupled to
fluorescein or Texas Red (Jackson), respectively. Images were prepared by means of a confocal microscope Leitz DM IRB (Leica, Bensheim, Germany) and
the graphic program ImageSpace (Molecular Dynamics; Sunnyvale, CA).
ACKNOWLEDGMENTS
We thank Ewa Barska and Birgit Kunkel for excellent
technical assistance during this project and Dr. Kerry
Tucker for critical reading of the manuscript. We gratefully acknowledge the gift of materials by Drs. H. Gu, K.
Rajewsky, J. Oberdick, M. Airaksinen, K. H. Herzog, J.
Morgan, C. Kellendonk, and G. Schütz. Many thanks for
the R1 cells to Andras Nagy and J. Roeder (University of
Toronto, Toronto, Canada).
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