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Divisions of Endocrinology (B.H., R.A.D., A.C.K.) and Nephrology (M.L.L.), Department of Medicine, Department of Oncology (M.P.), and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montréal, Québec, Canada H3T 1E2; and Calcium Research Laboratory (R.A.D., D.M., D.G.), Department of Medicine, Royal Victoria Hospital, McGill University, Montréal, Québec, Canada H3A 1A1
Address all correspondence and requests for reprints to: Andrew C. Karaplis, M.D., Ph.D., Lady Davis Institute for Medical Research, 3755 Côte Ste Catherine Road, Montréal, Québec, Canada H3T 1E2. E-mail: akarapli{at}ldi.jgh.mcgill.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The technology for producing such a conditional knockout is based on the cre/loxP site-specific recombination system of bacteriophage P1 that infects the bacterium Escherichia coli. Cre recombinase is an enzyme that catalyzes site-specific recombination between 34-bp sequences of phage DNA, termed loxP sites, thereby removing the DNA between them, leaving one loxP site behind (10). By combining the cre/loxP site-specific recombination system with embryonic stem (ES) cell technology, the capability of achieving conditional gene targeting has greatly expanded. The production of such a conditional knockout requires the generation of two mouse strains. One strain carries the gene of interest flanked by two loxP sites (floxed gene). The second is a conventional transgenic strain in which the Cre recombinase enzyme is expressed in a cell type- or developmental stage-specific manner. Appropriate mating between these two strains results in excision of the floxed DNA in a defined spatial or temporal manner.
In this study, we describe the generation of a mouse line in which loxP sites were introduced in the genome floxing nearly the entire coding region of the Pthrp gene. The capacity to excise the floxed gene in vivo was confirmed by crossing these animals to mice carrying the cre transgene driven by the human ß-actin promoter. Total body Cre-mediated recombination of the Pthrp gene resulted in a form of lethal chondrodysplasia, the characteristics of which faithfully recapitulated those of the conventional Pthrp knockout mouse.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To construct the remaining part of the targeting vector, ploxPneo-1 was digested with XhoI, the ends were blunted, and EcoRI linkers were attached. KpnI restriction of the resulting plasmid was followed by ligation of the 3.6-kb EcoRI fragment derived from the pPTHrPTV, composed of the 3'-flanking homology sequence, after blunting and addition of KpnI linkers. The 5'-flanking sequence of homology was derived as a 3.4-kb XhoI fragment from the pPTHrPTV plasmid and was inserted into the BamHI site of the resulting vector. Restriction of this plasmid with EcoRI released a DNA fragment encompassing both homology sequences, which when ligated to the NotI fragment derived from plasmid plox4/loxPGKneoNTRtkpA, resulted in the final targeting vector, pPTHrPfloxTV.
Generation of the Pthrp floxed mice
The pPTHrPfloxTV plasmid (25 µg) was linearized at the unique
NotI site and electroporated into R1 ES cells. Thirty-six
hours later, selection was initiated with 300 µg/ml G418, resistant
ES cell clones were isolated, and genomic DNA was prepared. Following
restriction with EcoRI and size fractionation on 0.8%
agarose gel, the DNA was transferred onto nitrocellulose filters and
hybridized with the 1.1-kb BamHI/SacI genomic
fragment containing sequences encoding exon 5 of the Pthrp
gene. One of the targeted clones underwent a second round of
electroporation with 25 µg of supercoiled plasmid pBS185 (from A.
Nagy, Lunenfeld Institute) containing the cre recombinase
gene under the control of human cytomegalovirus
promoter/enhancer. After selection in medium containing 2
µM ganciclovir for 5 days, clones were picked
and expanded. Genomic DNA was prepared and examined for type II
deletions by Southern blot analysis.
Appropriately targeted ES cells were microinjected into 3.5-day postcoitus BALB/c blastocysts and then transferred into uteri of 2.5-day postcoitus pseudopregnant CD1 mice. Seventeen days later chimeric animals were born. Extensively chimeric males were mated to BALB/c females and, following germ line transmission, animals heterozygous for the floxed allele were crossed to generate mice homozygous for the targeted allele.
Mouse strains
The Z/AP mice were provided by C. Lobe (Sunnybrook Health
Science Center, Toronto, Canada). The human
ß-actin-cre mice were a generous gift from B.
Morgan (Harvard University School of Medicine, Boston, MA) and G.
R. Martin (University of California, San Francisco, CA).
Histology
All animal studies were conducted in accordance with principles
and procedures dictated by the highest standards of humane animal care.
Newborn mice were killed, femurs, tibiae, and ribs were removed and
fixed in PLP fixative (2% paraformaldehyde containing 0.075
M lysine and 0.01 M sodium periodate solution)
for 24 h at 5 C. Samples were in turn decalcified in EDTA-glycerol
solution (14.5 g EDTA, 15 ml glycerol, 85 ml distilled water, and solid
sodium hydroxide added until a final pH of 7.3 was reached) for 1–2
days at 5 C. Following dehydration in graded alcohol, tissues were
embedded in low-melting-point paraffin, and 5-µm sections were cut on
a rotary microtome and stained with hematoxylin and eosin (H & E).
Preembedding lacZ staining
Preembedding lacZ staining was performed as
described, with some modifications (11). Samples were
fixed with PLP fixative overnight at 5 C, washed three times for 30 min
in lacZ wash buffer (2 mM
MgCl2, 0.01% sodium deoxycholate, 0.02%
Nonidet-P40 in PBS), and stained in 0.5 mg/ml X-gal, 5
mM potassium ferrocyanide, and 5
mM potassium ferricyanide in lacZ wash
buffer at 37 C overnight with shaking while protected from light.
Following staining, samples were decalcified, embedded in paraffin, and
5-µm sections were cut on a rotary microtome. Tissues were dewaxed,
hydrated by passage through graded alcohol series, washed in running
water for 3 min, and mounted with Kaiser’s glycerol jelly.
Human placental alkaline phosphatase staining
Tissue staining for human placental alkaline phosphatase
activity was performed as previously described (12).
Briefly, tissue sections were preincubated in TBS (50 mM
Tris-HCl, 150 mM NaCl, 0.01% Tween 20, pH 7.6) at 70-75 C
for 30 min to inactivate endogenous alkaline phosphatase activity.
Following overnight incubation in 1% MgCl2 and
100 mM Tris-maleate buffer (pH 9.2), sections were
incubated for an additional 2 h at room temperature in a
100-mM Tris-maleate buffer containing naphthol AS-MX
phosphate (0.2 mg/ml, Sigma, St. Louis, MO) dissolved in
ethylene glycol monomethyl ether as substrate and Fast Red TR (0.4
mg/ml, Sigma) as stain for the reaction product. After
washing with distilled water, the sections were counterstained with
Vector methyl green nuclear stain (Vector Laboratories, Inc., Ontario, Canada) and mounted with Kaiser’s glycerol
jelly.
Pthrp immunohistochemistry
Paraffin sections were stained for Pthrp using the
avidin-biotin-peroxidase complex technique. Sections were first treated
with 0.5% bovine testicular hyaluronidase (Sigma) for 30
min at 37 C, to increase antibody penetration and access to epitopes.
Rabbit antiserum against Pthrp 1–34 peptide was applied to sections
overnight at room temperature. As a negative control, the preimmune
serum was substituted for the primary antibody. After washing with high
salt buffer (50 mM Tris-HCl, 2.5% NaCl, 0.05% Tween 20,
pH 7.6) for 10 min at room temperature followed by two 10-min
washes with TBS, the sections were incubated with secondary antibody
(biotinylated rabbit antigoat IgG; Sigma), washed as
before and processed using the Vectastain ABC-AP kit
(Vector Laboratories, Inc.). Red pigmentation to demarcate
regions of immunostaining was produced by a 10- to 15-min treatment
with Fast Red TR/Naphthol AS-MX phosphate (Sigma,
containing 1 mM levamisole as endogenous alkaline
phosphatase inhibitor). The sections were then washed with
distilled water, counterstained with methyl green, and mounted with
Kaiser’s glycerol jelly.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The expectation was
that exon 4, when flanked by loxP sites, would remain fully
functional, but following Cre-mediated excision it would recapitulate
the Pthrp-null allele. In the design of the targeting
vector, the mouse phosphoglycerate kinase promoter was used to drive
expression of both the neor and
hsv-tk selectable genes. The ntr was inserted
between the two genes so that a bicistronic mRNA will be generated.
Because the ntr sequence includes an internal ribosomal
entry site (14), the hsv-tk gene would be
translated in a cap-independent manner.
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. G418-resistant ES cell
clones (276 clones) were isolated and genomic DNA was prepared and
examined by Southern blot analysis following restriction with
EcoRI and hybridization with probe A, a 1.1-kb
BamHI/SacI genomic fragment 3' to the targeted
homologous region and encompassing exon 5 of the murine
Pthrp locus (2). In the case of a targeted
recombination event, the wild-type allele would be expected to yield a
more than 20-kb fragment, whereas the floxed allele would result in an
11-kb fragment. Two clones having undergone the expected homologous
recombination event were identified and subjected to further
restriction mapping and Southern blot analysis to confirm the fidelity
of the targeting event using either an internal probe (probe B)
corresponding to a 382-bp AvrII/SmaI genomic
fragment encoding for part of exon 4 (13) or an
approximately 600-bp PCR-amplified fragment encompassing exon 3 of the
Pthrp gene (probe C), located 5' to the targeted homologous
region (Fig. 1A). In addition, the blots were stripped and rehybridized
with the neor probe, to verify that
only a single copy homologous integration event had occurred with no
evidence of random integration (data not shown).
One of the two targeted clones was expanded and the ES cells underwent
a second round of electroporation with 25 µg supercoiled pBS185
plasmid containing the cre recombinase gene under the
control of human CMV promoter/enhancer. The loss of the
neor-ntr-hsv-tk genes
cassette following excision by Cre recombinase activity, was expected
to make the ES cells ganciclovir resistant. After selection in medium
containing ganciclovir, 135 surviving clones were picked and expanded.
Genomic DNA was again prepared and examined for type II deletions (Fig. 1B; floxed exon 4 of Pthrp gene) by Southern blot analysis
following digestion with BamHI and hybridization with probe
B. The presence of a 5.2-kb band (type II recombination) in conjunction
with a 6.2-kb fragment (wild-type allele) in 5 of these clones
confirmed the successful removal of the selection genes cassette, while
leaving the floxed exon 4 of Pthrp intact. The presence of
the 9.5-kb mutant band along with the 6.2-kb wild-type band in several
clones indicated that selection with ganciclovir was not particularly
effective, perhaps due to low or no expression of the hsv-tk
gene.
Generating the floxed Pthrp mice
ES cells from one of these appropriately targeted clones were
microinjected into 3.5-day BALB/c blastocysts and chimeric male mice
were used to generate animals heterozygous for the floxed
Pthrp gene (Pthrp+/flox; +
and flox signify the presence of the wild-type and floxed alleles,
respectively). Offspring with this genotype were identified by Southern
blot analysis of tail genomic DNA and intercrossed to obtain mice
homozygous for the altered allele
(Pthrpflox/flox; Fig. 1C). As
expected, these animals were viable, fertile, and their overall
development appeared normal, indicative that the introduction of
loxP sites does not interfere with or alter to any
significant extent Pthrp gene expression in
vivo.
Analysis of Cre function in ß-actin-cre mice
Next, we wanted to determine whether Cre-mediated excision of the
floxed Pthrp allele could be successfully accomplished
in vivo. Total-body deletion of the Pthrp gene
was to be achieved using a mouse strain carrying a transgene where
cre was placed under the transcriptional control of
regulatory elements from the human ß-actin gene
(creactin), including the promoter, 5'
enhancer and intron, 3'-flanking untranslated region, and
polyadenylation sequences (15). The
creactin transgene was expected to be
expressed in all cell lineages of the early embryo. As a first step, we
set out to verify the in vivo efficacy of the
ß-actin promoter to drive the ubiquitous and functional
expression of Cre by crossing the
creactin mice to the double reporter
transgenic line Z/AP mice (11). The latter strain
expresses ubiquitously the lacZ reporter gene under control
of the CMV enhancer and chicken ß-actin
promoter before Cre-mediated excision takes place. However, when it
does occur, the lacZ gene that is floxed is removed,
permitting expression of the second reporter, the human placental
alkaline phosphatase gene (hPLAP), only in tissues that express Cre
recombinase. In this study, we sought to obtain offspring of the
genotype Z/AP;creactin, as determined
by lack of lacZ but presence of hPLAP staining of tail tips.
Figure 2 shows stained sections from soft
tissues and humeri from a Z/AP mouse as well as from a
Z/AP;creactin littermate. In the
absence of creactin, tissues failed to
stain for alkaline phosphatase but stained intensely for
lacZ activity, except for chondrocytes and adipocytes, as
was reported initially (11). However, in the presence of
the transgene, tissues stained intensely for alkaline phosphatase
activity, including chondrocytes and adipocytes, consistent with the
ubiquitous and complete excision of the floxed lacZ gene.
Therefore, we concluded that the cre gene in the
creactin mouse line functions
efficiently to excise floxed DNA segments in all cell lineages.
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). It was anticipated that these
animals would recapitulate the phenotypic changes observed in the
conventional knockout mouse (2). Therefore, we crossed
mice carrying the creactin transgene
to Pthrp+/- mice to obtain progeny of
the genotype
Pthrp+/-;creactin.
Matings of these animals with mice homozygous for the floxed allele
(Pthrpflox/flox) resulted in the
generation of progeny with the desired genotype,
Pthrp-/flox;creactin
(Fig. 3, B and C). These animals died in the perinatal period from
respiratory failure and exhibited all the phenotypic abnormalities
observed in the original Pthrp-null mice. As depicted in
Fig. 3D, Pthrp-/flox;creactin
mice had the characteristic chondrodystrophic features of domed skull,
shortened mandible resulting in protrusion of the tongue, narrow
thorax, protuberant abdomen, and shortened long bones. Skeletal
preparations stained with alcian blue (cartilage) and alizarin red S
(calcified tissue) confirmed the inappropriate and premature
ossification throughout the endochondral skeleton, and the anticipated
deformities that arise as a consequence of the complete absence of
Pthrp (data not shown). Histologic examination of long bones
from these animals validated the anticipated growth plate abnormalities
such as decreased size in the zone of proliferation, disorganization of
chondrocyte columns, and premature differentiantion of chondrocytes to
the hypertrophic state (Figs. 4, A–F,
and 5, A–F). Moreover,
immunohistochemical staining of mutant growth plates for Pthrp failed
to detect expression of the protein in chondrocytes (Fig. 6, A–D). These findings established that
the floxed Pthrp allele was accessible to Cre-mediated
excision in vivo. Subsequently, these studies were repeated
with mice of the
Pthrpflox/flox;creactin
genotype, and identical results were obtained, indicating that
Cre-mediated excision was equally effective in removing two copies of
the floxed allele.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The advent of gene targeting in ES cells has, over the last decade, revolutionized the in vivo study of gene function and has contributed enormously to our understanding of factors that modulate an ever-increasing number of physiological processes. Yet, as is often the case, early lethality of the mutant strain, an outcome exemplified by the Pthrp knockout mouse, or complex phenotypes preclude such studies at later stages of life or in specific tissues. These limitations have led to the development of a new generation of tools for controlling gene expression in vivo that aim to circumvent such problems associated with the conventional knockout technology. By combining the cre/loxP site-specific recombination with the homologous recombination technology, it has become possible to introduce genomic alterations that are restricting both spatially and temporally (16, 17).
Despite these advances, a number of potential drawbacks have become apparent with the advent of this novel technique. First, the ability to achieve tissue-specific gene deletion depends on the availability of cre transgenic mice that posses an exquisite degree of specificity and fidelity and achieve high levels in cre expression. Satisfactory tissue-specific restriction is achieved when Cre expression in these transgenic lines is under the transcriptional regulation of the appropriate tissue-specific promoter. On the other hand, nonspecific or low expression can severely confound interpretation of the resultant phenotype. Sorting out promoter specificity and activity has necessitated the generation of reporter mouse lines, like the Z/AP mouse, that provide a precise and accurate assay for Cre-mediated excisional activity at the cellular level (11, 18, 19). Although the Z/AP transgene was shown to be widely expressed in our studies, no lacZ expression was observed in chondrocytes and adipocytes, as originally reported (11). However, after Cre excision, both cell types stained intensely for hPLAP activity, suggesting that this discrepancy arises likely from possible sensitivity differences between the lacZ and the hPLAP reporters. In support of this conclusion is the profound alterations in the chondrocyte differentiation program observed in our loxP/cre progeny, confirming that Cre excisional activity had indeed taken place in these cells. Hence, the binary reporter system of the Z/AP transgene helps discriminate between lack of reporter expression and a lack of Cre excision.
The creactin mice used in the present study were particularly ideal for driving expression of the Cre protein ubiquitously, as required for achieving a total-body deletion of the Pthrp gene. In crosses using these mice, recombination of the target gene in the loxP/cre progeny has been reported to occur in every cell by the 64-cell stage of embryogenesis (15). The fact that Cre is expressed very early in development, leads to complete recombination in all cells of the embryo, as demonstrated in our studies following crosses of the creactin mice to the Z/AP reporter strain. This makes the creactin mice a very efficient line for generating progeny that carry the recombined form of the floxed allele.
Second, the resultant phenotype may be rather complex if deletion of the floxed gene is incomplete. This was a problem associated mainly with earlier studies that made use of the wild-type cre gene of P1 phage (20). The Cre recombinase expressed from the human ß-actin-cre transgene used in this study was exceptionally efficient in excising the floxed allele. This degree of efficiency arose from two modifications introduced in the cre gene: first, the sequences surrounding the ATG translation initiation codon matched those reported to be optimal for translation initiation in eukaryotic cells (21), and second, the coding sequence for the seven-amino acid nuclear localization signal of the large T antigen of SV40 had been introduced into the amino-terminal region of the cre open reading frame (22).
These potential limitations notwithstanding, studies are now underway aiming to target the floxed Pthrp allele in a tissue-specific fashion. Crossing the Pthrpflox/flox strain to mice exclusively expressing Cre in a variety of cell types will facilitate the functional analysis of Pthrp’s role in the postnatal state. Findings arising from such studies will undoubtedly provide us with otherwise unobtainable information about potential actions of Pthrp in adult bone homeostasis, mammary gland, prostate and pancreatic islet cell function, blood pressure control and vascular responsiveness, and neuronal protection from apoptotic cell death.
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Footnotes |
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2 Recipients of Medical Research Council of Canada/Canadian
Institutes of Health Research Doctoral, Fellowship, and Scientist
Awards, respectively.
3 Chercheur-Boursier Clinicien of the Fonds de la Recherche en
Santé du Québec.
Received October 27, 2000.
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,
loxP sites; ntr, the 5' ntr of
encephalomyocarditis virus; Pgk-1, the mouse
phosphoglycerate kinase promoter and polyadenylation signal
(pA). B, Cre-mediated excision of the selectable marker
genes cassette. Transient transfection of one targeted ES clone with
plasmid expressing Cre under the control of the CMV
promoter. Below, three potential recombination events (types I, II, and
III) are illustrated but only clones with type I and II deletions are
expected to survive ganciclovir (2 µM) selection. Genomic
DNA from several surviving clones was restricted with
BamHI and resulting fragments (double
arrowhead lines) probed with probe B. C,
ES cells with type II deletion were used to generate the
Pthrpflox/flox mice using standard
protocols. Shown in this study, is a Southern blot of tail tip genomic
DNA following digestion with BamHI and hybridization
with probe B from wild-type (+/+) mice and litter mates heterozygous
(+/flox) and homozygous (flox/flox) for the floxed Pthrp
allele.
