TECHNOLOGY REPORT

Transgenic Mice That Express Cre Recombinasein Hypertrophic ChondrocytesGuan Yang,1 Fang Cui,1 Ning Hou,1 Xuan Cheng,1 Jishuai Zhang,1 Youliang Wang,1

Nan Jiang,1 Xiang Gao,2 and Xiao Yang1*1Genetic Laboratory of Development and Diseases, Institute of Biotechnology, Beijing, P.R. China2Model Animal Research Center, Nanjing University, Nanjing, P.R. China

Received 25 September 2004; Accepted 16 February 2005

Summary: In order to investigate the physiological con-trol of hypertrophic chondrocytes which present the ter-minally differentiated form of chondrocytes, we gener-ated a mouse line expressing the Cre recombinaseunder the control of the mouse type X collagen (Col10a1)promoter. In situ hybridization analysis demonstratedthe expression of Col10a1-Cre transgene in hypertro-phic chondrocytes of femur at postnatal day 2 (P2). Inorder to test the excision activity of the Cre recombi-nase, the Col10a1-Cre transgenic line was crossed withthe mouse strain carrying the Smad4 conditional alleles(Smad4co/co) and the reporter line ROSA26. Multipletissue PCR of Col10a1-Cre;Smad4co/þ mice revealed therestricted Cre activity in tissues containing hypertrophicchondrocytes. LacZ staining revealed that the Cre activ-ity was observed in the cartilage primordia of ribs atE14.5 and only detected in the lower hypertrophic regionof ribs at P1. These data suggest that the Col10a1-Cremouse line described here could be used to achieveconditional gene targeting in hypertrophic chondro-cytes. genesis 42:33–36, 2005. VVC 2005 Wiley-Liss, Inc.

Key words: hypertrophic chondrocyte; Cre recombinase;transgenic mouse; growth plate

Endochondral ossification, which is the major processleading to the development of most of the mammalianskeletons, involves temporally and spatially balancedcycles of proliferation, maturation, hypertrophy, and cal-cification of chondrocytes (Karsenty et al., 2002; Kro-nenberg, 2003). Hypertrophic chondrocytes that arecomposed of two subpopulations have been consideredthe principal engine of bone growth. The prehypertro-phic chondrocytes line up right below the proliferatingchondrocytes and express Ihh (St-Jacques et al., 1999),FGFR1 (Wang et al., 2001) and Cbfa1 (Otto et al.,1997). The hypertrophic chondrocytes characterized bythe expression of type X collagen are larger than the pro-liferating and prehypertrophic chondrocytes and will diethrough apoptosis (Gibson et al., 1995). Hypertrophicchondrocytes change the composition and properties ofsurrounding matrix, allow invasion of blood vessels thatbring in osteoblast progenitors, and direct adjacent peri-

chondrial cells to become osteoblasts and form a bonecollar (Kronenberg, 2003). Up to now, very little isknown about the genetic control of the functions ofhypertrophic chondrocytes. Identification and analysisof the genes that are involved in the above-mentionedprocesses can help to better understand the physiologi-cal functions of hypertrophic chondrocytes. The Cre-loxP system provides us with a powerful means to ena-ble cell- or tissue-deletion of a targeted gene in specifictissues of interest (Rossant et al., 1999), including thehypertrophic chondrocytes.

Type X collagen is the only known hypertrophic chon-drocyte-specific molecular marker (Kwan et al., 1997).It has been reported that the –120 bp to þ1 bp fragmentnear the start site of transcription is the likely basal pro-moter (Beier et al., 1996). Although some other mole-cules such as BMP-6 (Grimsrud et al., 1999), alkalinephosphatase (Vaananen, 1980), or osteopontin (Gersten-feld et al., 1996) are also highly expressed within thezone of hypertrophy compared to the other chondrocyteregions, their relatively wide expression patterns inother tissues have limited their applications to ourresearch.

In the current study, we generated a transgenic mouseline (Col10a1-Cre) in which the Cre recombinaseexpression is driven by the 1.0 kb promoter of themouse type X collagen gene (see Materials and Meth-ods). To determine the spatial expression pattern of theCre recombinase in the transgenic mouse, we performedin situ hybridization. Cre transcripts were only detectedin the lower hypertrophic zone of postnatal day 2 (P2)

* Correspondence to: Xiao Yang, Ph.D., Institute of Biotechnology, 20

Dongdajie, Fengtai, Beijing 100071, P.R. China.

E-mail: yangx@nic.bmi.ac.cn

Contract grant sponsor: National Science Fund for Distinguished Young

Scholars; Contract grant numbers: 30025028, 30128013; National Natural

Science Foundation of China; Contract grant number: 30430350; National

Gongguan Projects; Contract grant number: 2001BA710B.Published online in

Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/gene.20120

' 2005 Wiley-Liss, Inc. genesis 42:33–36 (2005)

femur (Fig. 1a,b). To further check the tissue distributionand the excision activity of Cre recombinase, theCol10a1-Cre transgenic line was crossed with a mousestrain that carries Smad4 conditional alleles(Smad4co/co) (Yang et al., 2002). Cre-mediated excisionof exon 8 of Smad4 in different tissues isolated from theCol10a1-Cre;Smad4Co/þ offspring was evaluated byPCR. PCR analysis of heart, liver, spleen, lung, kidney,intestine, stomach, pancreas, trachea, skin, and skeletaltissues revealed that the Cre mediated recombinationoccurred in all tested cartilage tissues containing hyper-trophic chondrocytes (Fig. 2b). The Cre activities werealso detected in skin tissues (Fig. 2b).

To visualize the Cre activity and the excision effi-ciency, we crossed the Col10a1-Cre transgenic line witha reporter mouse line ROSA26 that will express LacZ intissue where Cre-mediated recombination occurs (Sor-iano, 1999). We compared the X-gal staining betweenCol10a1-Cre;ROSA26 and ROSA26 littermates at E14.5and P1. LacZ activity was detected in cartilage primordiaof E14.5 ribs where type X collagen expression was alsodetected (Fig. 3a,b). Skin tissue also showed Cre activity,while lung, liver, and other tissues tested did not expressthe Cre recombinase (Fig. 3c,d; data not shown). StrongLacZ staining was observed in the lower zone of hyper-trophy of Col10a1-Cre;ROSA26 mice at P1 (Fig. 3e), indi-cating the clonal expression of Cre recombinase in thehypertrophic chondrocytes. Although a few LacZ-posi-tive cells were found in perichondria (Fig. 3e,f, arrow-head) and on the trabecular surface in both Col10a1-Cre;ROSA26 and ROSA26 mice, none of the hypertro-phic chondrocytes in ROSA26 was stained blue (Fig. 3f).The Cre activity was not detected in resting chondro-cytes and proliferating chondrocytes (Fig. 3e,f), in agree-ment with the in situ hybridization results (Fig. 1b). Alto-gether, these data indicated that Cre recombinase wasexpressed specifically in lower hypertrophic chondro-cytes in the Col10a1-Cre transgenic mice at a levelallowing recombination at the ROSA26 and other loci.

In conclusion, we generated a transgenic mouse linethat express Cre recombinase specifically in lowerhypertrophic chondrocytes, which will enable the dis-ruption of loxP flanked genes in these cells. Previousstudies have shown that 4-kb and 4.6-kb Col10a1 pro-moters can direct reporter expression selectively tolower hypertrophic chondrocytes in transgenic mice(Zheng et al., 2003; Gebhard et al., 2004), our resultsindicated that the 1.0 kb promoter of the type X collagengene we used here might include the important cis regu-latory elements which can specifically drive the expres-sion of type X collagen within hypertrophic chondro-cytes. Our results also suggested that further regulatoryelements in the Col10a1 gene should be included todirect the expression of Cre in all hypertrophic chondro-cytes. Although the Cre activity was also found in skintissue, there is no consequence if the floxed gene is notexpressed in this tissue. Nevertheless, the established

FIG. 2. a: Schematic diagram of the floxed Smad4 alleles pre- andpost-Cre-mediated recombination. A 234-bp fragment could beamplified from the deleted Smad4 allele by PCR using primers 5and 6 (P5/P6) after the Cre-mediated excision of exon 8 of Smad4.b: Tissue distribution of Cre activities. A 234-bp fragment wasamplified from tissues with Cre activities. A 481 bp fragment wasamplified using primers 3 and 4 from all tissues that carried theCol10a1-Cre transgene in the genome. Tissue types were as indi-cated. Cartilage (T), cartilage from tibias; cartilage (H), cartilage fromhumeri; cartilage (R), cartilage from ribs; skin (D), skin tissue fromdorsal side; skin (A), skin tissue from auricle.

FIG. 1. Expression of Cre mRNA inhypertrophic chondrocytes of Col10a1-Cre transgenic mice. In situ hybridizationof tissue sections from P2 femur ofCol10a1-Cre transgenic mouse (a,brightfield; b, darkfield) demonstratedthat Cre transcripts were only detectedin the hypertrophic chondrocytes whichlined up below the prehypertrophicchondrocytes. No signal was detected inchondrocytes of wildtype control (c).

34 YANG ET AL.

Col10a1-Cre transgenic line will be a valuable tool forstudying the genetic regulation of hypertrophic chon-drocyte function.

MATERIALS AND METHODS

Generation of the Col10a1-cre Transgenic Mice

A 1.0 kb promoter of type X collagen gene was obtainedfrom mouse genomic DNA using PCR primer 1 (50-GGTACC TAA GGA TAT TTC TGT AAG GCT GTG AA-30) andprimer 2 (50-GTC GAC GAG ATG CGT GGG GCA GCT T-30). Two restriction sites (GGT ACC for KpnI and GTCGAC for SalI) were added into the 50 and 30 of the pro-moter sequence by PCR mutagenesis, respectively. Thispromoter was then inserted into the cloning sites (KpnI/SalI) of an engineered vector containing Cre codingregion and hGH polyadenylation signal (Postic et al.,1999). The final Col10a1-Cre transgenic construct con-sisted of 50 sequence of 1.0 kb of the Col10a1 promoterregion and 1.2 kb of Cre coding region followed by the2.1 kb of hGH polyadenylation signal.

The 4.3-kb linearized insert was excised from the vec-tor backbone by KpnI-XbaI digestion and purified usingQIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA).The inserts were microinjected into the pronuclei of fer-tilized Kunming mouse oocytes to generate the trans-genic mice. Founders were preliminarily detected by

PCR on tail genomic DNA using primer 3 (50-GCC TGCATT ACC GGT CGA TGC-30) and primer 4 (50-CAG GGTGTT ATA AGC AAT CCC-30). Southern analysis of tailDNA was taken to confirm the results of PCR.

In Situ Hybridization

In situ hybridization was performed using standard pro-cedures. Sense and antisense probes for Cre were gener-ated from the pGEM-T Easy Vector carrying the SalI-EcoRI fragment of the Cre gene. Probes were labeledwith 35S-UTP using the MAXIscript in vitro transcriptionkit (Ambion, Austin, TX). Slides were dipped in emulsion(Amersham Pharmacia, Piscataway, NJ) and exposed for10 days before developing.

Tissue Distribution of the Cre Transgene

Mice that were homozygous for the ‘‘floxed’’ Smad4allele (Smad4Co/Co) (Yang et al., 2002) were crossedwith the Col10a1-Cre transgenic mice. Genomic DNAswere isolated from multiple tissues of P2 Col10a1-Cre;Smad4co/þ mouse. Cartilage tissues were taken fromgrowth plates of tibias and ribs under a dissecting micro-scope (Nikon). Primers 3 and 4 that amplified a fragmentof 481 bp were used to detect the existence of Cre gene.In addition, primer 5 (50-CCT TAG TTG AAG CTT ATAACT TCG-30) and primer 6 (50-GAC CCA AAC GTC ACC

FIG. 3. Cre activity in Col10a1-Cre trans-genic mice was visualized by X-gal stainingon sections from E14.5 and P1 Col10a1-Cre, ROSA26 double transgenic (a,c–e),and ROSA26 mice (f). LacZ activity wasdetected in cartilage primordia of E14.5 ribs(a). In situ hybridization with Col10a1 on aparallel section is shown (b). LacZ stainingwas observed in skin tissue (c) but not inlung (d). Strong LacZ staining was detectedin the lower hypertrophic zone (e) of rib.Arrowheads in e and f point to the nonspe-cific stained perichondrial cells. In situhybridization with Col10a1 on a parallel sec-tion of the rib is shown (g). Pc, proliferatingchondrocytes; preHc, prehypertrophic chon-drocytes; Hc, hypertrophic chondrocytes.

35TRANSGENIC MICE THAT EXPRESS CRE RECOMBINASE

TTC AC-30) were used to amplify a fragment of 234 bpfrom the Smad4 allele after the Cre mediatedrecombination.

X-Gal Staining

P1 Col10a1-Cre;ROSA26 double transgenic pups werefixed in 0.2% glutaraldehyde in 0.1 M phosphate buffer(pH 7.3) for 5 h at 48C. They were then rinsed in 0.1 Mphosphate buffer containing 2 mM MgCl2, 0.2% NP-40,and 0.1% Na-deoxycholate. The staining was carried outin the washing buffer supplemented with 1 mg/ml X-gal,6 mM potassium ferrocyanide, and 6 mM potassium ferri-cyanide for 5 h. Pups were subsequently embedded inparaffin, sectioned, and counterstained with NuclearFast Red.

ACKNOWLEDGMENTS

We thank Chuxia Deng for Smad4 conditional gene tar-geting mouse. We also thank Xiaohong Tan for helpfuldiscussion.

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