Introduction Cartilage–bone transition by endochondral ossification is a highly complex process which determines not only longitudinal growth of long bones, ribs and vertebrae, but also plays a critical role in bone fracture callus healing, osteophyte formation and cartilage tissue engineering. Decisive steps in this process are proliferation, maturation and hypertrophy of chondrocytes in the growth plate which play a key role in mineralization, apoptosis and induction of resorption of hypertrophic cartilage by invading bone marrow. These events are regulated by synergistic action of several signaling pathways and transcriptions factors controlled by a multitude of growth factors and hormones. 1998 2000 2002 1998 1998 2001 1996 2005 2004 2002 2005 2006 2005 2002 1997 1998 1999 2000 1999 2000 2003 1994 2001 2002 2004 2001 2001 2003 1998 2001 2000 2002  LacZ 2004 2003 2004 lacZ LacZ lacZ E. coli 2000 2001 lacZ cis Materials and methods BAC clones http://www.genome.ucsc.edu/ 2002 2004 : GGG AAT TCT TAC CTT AAA GTA GAT ACA TG (amplicon size 222 bp). Construction of targeting vector placH+Col10a1-lacZ-frt-neo-frt 1 Sal Nco Sal Nco Sal Nco 1995 lacZ Nco lacZ Fig. 1 lacZ a gray boxes black b E. coli c  Hin Xho 2000 2001 TA ACT GAT CGC GGC CAG CTT GAA GTT Hin T TGT GTC TTG GGG CTA GC AAG TGG GCC CTT TAT GCC TGT GGG CGT TTG GTA CCG TT C TAT TCC AGA AGT AGT GAG GAG GCT TT Hin Homologous recombination and detection of BAC transgene E. coli cl BAD flpe exo, bet, gam 2001 Xho Xho E. coli 1988 2001 2000 2001 BAC DNA purification and generation of transgenic mice E. coli 2001 2 PIS 2005 2005 Fig. 2 PIS 3 Not 4 Pme 5 Nru 6 1 2 7 PIS  Genotyping of transgenic mouse lines Transgenic founder animals were selected by PCR of tail biopsies using primers pairs P1/P2 and P3/P4 (see above) detecting recombination events. Each sample was standardized by PCR using P1/P4 primer pair which generates a 538 bp fragment from the wt Col10a1 allele. Founders derived from injections of F1 hybrids were crossed with C57BL6, whereas founders derived from FVB oocytes were crossed with FVB mice. For genotyping of newborn mice and embryos DNA was extracted from skin or placenta, and transgene DNA was identified by PCR as above. Histological analysis and in situ hybridization 2 1995 1992 32 2006 Determination of transgene copy number by Southern hybridization Bam Bam 32 Results  Construction of a placH-Col10a1-lacZ-frt-neo-frt targeting vector and homologous recombination lacZ lacZ Nco 1 E. coli  Xho 1 1 Materials and Methods 1 Materials and Methods 2 Generation of transgenic mice PISceI lacZ 1 3 1 Fig. 3 a Bam 32 1 b  Table 1 Materials and methods Founder # Strain sex β-gal activity BAC copy no. 800 FVB, m + 5 986 FVB, f + 3 1501 FVB, f + 1 1504 FVB, m + 3 1508 F1, f ND 2 1510 F1, m ND 1 1515 F1, f ND 4 1516 F1, f + 6 1520 F1, m + 7 1524 F1, f ND 2 1527 F1, m ND 1 1533 FVB, m ND 1 1534 FVB, m + 4 ND 4 5 6 3 3 Fig. 4 a b e a b c s h f) (cl) c1, c2 p c d b arrow n d  Fig. 5 a b c i a b c d e C1  eb h m  MC mh r f g h i  Fig. 6 a e g a b c d e f h g  Specific expression of the BAC-Col10a1-LacZ-neo transgene in hypertrophic cartilage BAC-Col10a1-lacZ-neo 4 4 lacZ 5 6 6 4 4 4 4 4 5 4 5 4 5 4 5 5 5 6 6 Discussion lacZ 2004 2003 1995 1997 2002 2002 1997 2004 2004 2004 1996 2003 lacZ 2004 lacZ 2004 1992 1991 2000 2006 2000 2003 Most E14.5- to E16.5-day BAC-Col10a1-lacZ-neo transgenic embryos showed some X-gal staining in hair follicles of the forehead and snout, and of the epidermis of the tail, legs and shoulder, which disappears after birth. In the X-gal positive spots, staining was restricted to the basal layer of keratinocytes. Since these cells do not express type X collagen, the effect may be due to endogenous galactosidase activity, or a result of LacZ activation by other, Col10a1-unrelated regulatory elements located in the BAC clone. In addition to Col10a1, the BAC clone RP23-192A7 contains coding regions of three additional genes Tspyl-1, Tspyl1-4 and Nt5dc1. Whether these genes are expressed under their endogenous promoters in the BAC transgenic mice is currently investigated. If they are expressed, they could affect the phenotype of the established transgenic mouse lines, but no phenotypic alteration in skeletal elements nor in other tissues have been observed in transgenic embryos, newborn or adults as compared to wildtype littermates. The high intensity of X-gal staining under the control of the BAC-Col10a1 promoter allowed the visualization of discrete zones of Col10a1 expression in hypertrophic cartilage of anatomical structures not investigated so far in such detail and completeness, for example the skull or the inner and outer aspects of the thoracal and cervical vertebrae. The data presented in this study illustrate the superior potential of reporter gene expression analysis in combination with BAC recombineering technique as compared to conventional in situ hybridization analysis. lacZ lacZ