Minerva Access is the Institutional Repository of The University of Melbourne
Author/s:
GOLUB, SUZANNE; ROGERSON, FRASER; FOSANG, AMANDA; EAST, CHARLOTTE
Title:
Abundant LacZ activity in the absence of Cre expression in the normal and inflamed
synovium of adult Col2al-Cre; ROSA26R(Lacz) reporter mice
Date:
2013-02-01
Citation:
Fosang, AJ; Golub, SB; East, CJ; Rogerson, FM, Abundant LacZ activity in the absence of
Cre expression in the normal and inflamed synovium of adult Col2al-Cre; ROSA26R(Lacz)
reporter mice, OSTEOARTHRITIS AND CARTILAGE, 2013, 21 (2), pp. 401 - 404 (4)
Persistent Link:
http://hdl.handle.net/11343/44056
Accepted Manuscript
Abundant LacZ activity in the absence of Cre expression in the normal and inflamed
synovium of adult Col2a1-Cre;ROSA26RLacZ reporter mice
Amanda J. Fosang, Suzanne B. Golub, Charlotte J. East, Fraser M. Rogerson
PII:
S1063-4584(12)01026-6
DOI:
10.1016/j.joca.2012.11.013
Reference:
YJOCA 2788
To appear in:
Osteoarthritis and Cartilage
Received Date: 9 August 2012
Revised Date:
22 November 2012
Accepted Date: 26 November 2012
Please cite this article as: Fosang AJ, Golub SB, East CJ, Rogerson FM, Abundant LacZ activity in the
absence of Cre expression in the normal and inflamed synovium of adult Col2a1-Cre;ROSA26RLacZ
reporter mice, Osteoarthritis and Cartilage (2012), doi: 10.1016/j.joca.2012.11.013.
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Abundant LacZ activity in the absence of Cre expression in the normal and
inflamed synovium of adult Col2a1-Cre;ROSA26RLacZ reporter mice
Amanda J. Fosang, Suzanne B. Golub, Charlotte J. East & Fraser M. Rogerson
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University of Melbourne Department of Paediatrics and Murdoch Childrens Research
Institute, Royal Children’s Hospital, Parkville, Victoria 3052, Australia
Brief reports should not exceed an abstract of 250 words, a main text of 2000 words, 15
references, and 2 figures and/or tables.
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Brief Report
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Key words: Col2a1, Col2a1-Cre, Cre recombinase, synovium, inflammatory arthritis
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Competing Interests: The authors have no competing interests to declare.
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ABSTRACT
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Recent analyses of Col2a1-Cre;ROSA26R reporter mice showed that synovial fibroblasts in
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7-day mice were LacZ positive, due to a history of Col2a1-Cre expression conferred by their
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origin in the interzone of the developing joint. We have examined LacZ staining in adult
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Col2a1-Cre+/0;ROSA26RLacZ mice, with and without inflammatory arthritis, and found that
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synovial fibroblasts in normal and inflamed synovium are LacZ positive, but Cre negative.
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Our results suggest that Cre-mediated recombination in joint interzone cells during
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development endure in adult synovial cells despite the absence of ongoing Cre expression.
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These findings have important implications and applications for the study of synovial
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inflammation in models of experimental arthritis.
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INTRODUCTION
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Our interest in the source of destructive proteinases in synovial joints directed our attention to
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the Col2a1-Cre mouse for the purpose of ablating proteinase activity in chondrocytes, but not
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synovial fibroblasts. In the Col2a1-Cre transgenic mouse, Cre recombinase is expressed
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under the control of the type II collagen (Col2a1) regulatory region [1, 2]. When crossed with
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the ROSA26 reporter mouse carrying the bacterial ß-galactosidase gene flanked by LoxP sites
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[3, 4], successful recombination yields mice expressing ß-galactosidase (LacZ) activity in
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cells that express, or whose precursor cells have previously expressed, Cre recombinase.
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Previous studies with Col2a1-Cre mice have shown that although there is a low level of Cre
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expression in notochord, brain, spinal cord, eye, heart and epidermis, Col2a1-Cre expression
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is largely restricted to chondrocytes and presumptive joint cells [1]. However, more recent
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studies have revealed LacZ activity in synovium of 7-day Col2a1-Cre+/+;ROSA26RLacZ mice
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[5]. These studies showed that although 7-day synovial fibroblasts were negative for type II
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collagen immunoreactivity, they had a history of Col2a1-Cre expression due to their origin in
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the interzone of the developing joint [5, 6]. As a consequence, the genetic modifications
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caused by Cre-mediated excision of floxed alleles in interzone cells during embryonic
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development were inherited by daughter cells resident in the synovium in later generations. In
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subsequent work the same authors showed that during development, cells with a history of
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Col2a1 expression combined with invading cells that are Col2a1-negative, to form the
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medial meniscus [7].
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The fate of adult synovial cells in Col2a1-Cre transgenic mice has not been examined,
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despite the utility of this mouse for studies of inflammatory arthritis. Accordingly, it is
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unclear whether cells of the adult synovium descend entirely from interzone-derived Col2a1-
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Cre expressing cells, in which case all cells of the adult synovium will have a history of
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Col2a1-Cre expression, or whether synovial cells present at 7-days represent a sub-
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population of cells that are present during the early post-natal period, but are gradually
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replaced by invading cells with no history of Col2a1-Cre expression. This is a question of
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critical importance for investigators seeking to achieve cartilage-restricted gene expression in
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models of inflammatory arthritis.
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We have crossed the Col2a1-Cre mouse with the ROSA26R reporter mouse and examined
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LacZ activity in adult mouse synovium. We found that although the Cre transgene was not
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expressed in synovial cells in the mouse knee, many synoviocytes were nevertheless LacZ
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positive, indicating that these cells have a history of Col2a1-Cre expression. This finding is
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especially relevant for models of experimental arthritis that induce synovial hyperplasia and
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inflammation.
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METHODS
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After mating Col2a1-Cre0/0 mice [1] with ROSA26RLacZ mice [3], the F1 progeny were mated
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with ROSA26R
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homozygous for the ROSA26RLacZ allele but negative for the Col2a1-Cre allele were used as
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negative controls.
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mice to generate Col2a1-Cre+/0;ROSA26RLacZ F2 progeny. Mice
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LacZ
LacZ expression was detected by X-gal staining [3]. Healthy knee joints dissected from
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Col2a1-Cre+/0;ROSA26RLacZ and control mice at 2, 4 and 8 weeks of age were fixed in 4%
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paraformaldehyde (PFA) in PBS for 2 h at 4°C, and decalcified in 7% (w/v) EDTA in PBS
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for 14 days at 4°C. The joints were then incubated in 30% sucrose for 24 h at 4°C and
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embedded in OCT. Cryosections (10 µm) were incubated overnight at room temperature,
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then overnight at 37°C with X-Gal solution (20mM Tris-HCl, pH 7.5, containing 1 mg/mL
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X-Gal, 0.25mM potassium ferrocyanide) then counterstained with Nuclear Fast Red. For
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immunohistochemistry, the anti-Cre antibody (1:2,000) was from Novagen and the swine
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anti-rabbit-HRP-conjugated immunoglobulin (1:100) was from Dako.
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RESULTS
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LacZ activity was detected in knee joints from Col2a1-Cre+/0;ROSA26RLacZ mice at 2, 4 and 8
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weeks of age (Figure 1 A, B, D). As expected, LacZ activity was present in articular (AC)
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and growth plate (GP) chondrocytes at all ages, and in the meniscus (M). LacZ activity was
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also present in cells of the synovial layer of the knee joint (Figure 1 insets, arrowed). No X-
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gal staining was detected in Cre-negative control mice (Figure 1C).
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The finding of X-gal staining in the normal synovium prompted us to examine LacZ
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activity in Col2a1+/0;ROSA26RLacZ mice with antigen-induced arthritis (AIA), which is
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characterised by extensive synovial fibroblast hyperplasia. AIA was induced as described
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previously [8] and approved by the Animal Ethics Committee of the Murdoch Childrens
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Research Institute. Briefly, on day 0, 8-week old mice were primed with an intradermal
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injection of 50µg methylated BSA (mBSA) in Freund’s Complete Adjuvant at two sites near
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the base of the tail. Arthritis was induced on day 7 by an intra-articular injection of 10µL of
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20mg/mL mBSA directly into both knees. The joints were harvested at day 14, fixed, then
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processed for cryosectioning. X-gal staining revealed LacZ activity in synovial fibroblasts
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throughout the inflamed synovium (Figure 2A). At high magnification, large numbers of X-
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gal-negative neutrophils and mononuclear cells were also visible in the inflamed synovium
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(Figure 2B). There was no X-gal staining in sections from Cre-negative control mice (Figure
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2C-D). X-gal staining in trabecular and cortical bone and the perichondrium (Figure 2A) is
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consistent with Cre activation at early stages of skeletal development in both chondrocytic
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and osteoblastic cell lineages [1] and could also be due to endogenous galactosidase activity
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present in postnatal bone.
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To confirm that X-gal staining was not due to existing Cre activity in the inflamed synovium,
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we next analysed tissue sections for the presence of Cre protein using an anti-Cre antibody.
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The results showed that while Cre protein was readily detected in the growth plate cartilage,
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there was no Cre immunoreactivity detected in the synovium (Figure 2E). These results
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show that fibroblasts in the inflamed synovium of adult Col2a1-Cre+/0;ROSA26RLacZ mice
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express LacZ activity, not because the Cre transgene is expressed in adult synoviocytes, but
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because the synovial cells have a history of Col2a1-Cre expression [7] that causes ongoing
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expression of the Cre-recombined ROSA26RLacZ allele in the absence of Cre recombinase.
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There was no Cre immunostaining in sections from Cre-negative control mice (Figure 2F).
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DISCUSSION
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The restricted expression of type II collagen in chondrocytes suggested that Col2a1-Cre
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transgenic mice crossed with mice expressing floxed alelles could be used to monitor the
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specific consequences of Cre-mediated recombination in a chondrocyte-specific manner.
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However, we show here that the Col2a1-Cre mouse is not solely suited to this purpose,
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because Cre recombinase is expressed at early developmental stages in interzone cells that
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ultimately give rise to synovial fibroblasts in the adult mouse. Since other studies in mouse
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[7] and chick embryos [9] have shown that cells in peri-joint sites migrate into the developing
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joint to replace resident cells, it was important to establish whether cells in the adult mouse
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synovium might also arise from pericellular sites with no history of Col2a1 expression. Our
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results herein show that this is not the case.
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This brief report redefines the usefulness of the Col2a1-Cre mouse and shows that
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recombined genes are expressed in both chondrocytes and synovial fibroblasts. If
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chondrocyte-specific recombination is required then one strategy for avoiding recombination
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of floxed genes in interzone cells during development is to induce Col2a1-Cre activity post-
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natally using, for example, Col2a1-CreER transgenic mice that express Tamoxifen-dependent
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Cre-recombinase [10, 11]; this allows for the temporal control of Cre expression.
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In summary, our findings expand the utility of the Col2a1-Cre mouse. We conclude that the
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Col2a1-Cre mouse is a reliable and unique resource for manipulating gene expression in the
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articular compartment, not only in chondrocytes, but also in synovial fibroblasts, and as a
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consequence, we emphasise that the hyperplastic nature of the inflamed synovium requires a
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new interpretation of the results from inflammatory arthritis experiments in Col2a1-Cre
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transgenic mice.
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FIGURE LEGENDS
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Figure 1: LacZ activity in Col2a1-Cre+/0; ROSA26RLacZ mice at 2 weeks (n=3)(A), 4 weeks
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(n=5)(B) and 8 weeks (n=5)(D) of age. LacZ activity is absent in the negative control mice
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(Col2a1-Cre0/0; ROSA26RLacZ) at all ages; 4 weeks of age is shown in panel C (n=2). LacZ
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activity is prominent in chondrocytes of the articular cartilage (AC), growth plate cartilage
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(GP) and meniscus (M). Insets are higher power views of the synovium, showing blue
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staining in synovial fibroblasts (arrowed). Bar = 200µm.
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Figure 2: LacZ activity but no Cre expression in Col2a1-Cre0/+;ROSA26RLacZ mice in the
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AIA model of inflammatory arthritis (n= 4)(A,B). LacZ activity is absent in the Col2a1-
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Cre0/0;ROSA26RLacZ negative control mice (n=3)(C,D). Panel B is a higher power view of
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panel A, showing extensive X-gal staining in the hyperplastic synovium, but also unstained
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inflammatory cells. Cre immunoreactivity is present in the Col2a1-Cre-/+;ROSA26RLacZ
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growth plate but absent in the synovium (E). No Cre staining was detected in joints from the
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negative Cre negative control mice (F) Bar (A,C) = 1mm, (B,D) = 100µm, (EF) = 500µm.
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Acknowledgments
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The authors thank Dr Atilla Aszodi for the Col2a1-Cre mouse, Professor Andrew Sinclair for
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sharing the ROSA26RLacZ mouse and Dr Amanda Notini for genotyping the LacZ allele. This
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work was supported by research funding from the National Health & Medical Research
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Council (Australia) and by the Victorian Government's Operational Infrastructure Support
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Program.
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Contributions
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All authors contributed to
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1) The conception and design of the study, or acquisition of data, or analysis and
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interpretation of data
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2) Drafting the paper or revising it critically for intellectual content
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3) Final approval of the submitted version of the manuscript
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