Specific expression of Cre recombinase in hypertrophic cartilage under the control of a BAC-Col10a1 promoter

Abstract

Previously we have shown that insertion of a LacZ reporter gene into the Col10a1 gene in the context of a bacterial artificial chromosome (BAC) drives strong and specific expression of LacZ in hypertrophic cartilage of transgenic mice [Gebhard S., Hattori T., Bauer E., Bosl M.R., Schlund B., Poschl E., Adam N., de Crombrugghe B., von der Mark K., 2007 Histochem. Cell Biol. 19 127:183-194]. BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter. Here we report on the generation of Col10a1-specific Cre deleter mice using a BAC recombineering technique based on homologous recombination in E. coli. Sixteen BAC-Col10-Cre transgenic lines were generated containing between 1 and 5 copies of the BAC-Col10-Cre gene. All lines tested so far expressed Cre specifically in hypertrophic chondrocytes of E16.5 and P1 growth plates of long bones, ribs, vertebrae and sternum as examined by crossing with ROSA26 reporter mice. Cre activity was detected as early as E13.5 when hypertrophic cartilage develops in the diaphysis of femur and humerus. The data confirm that expression of Cre under the control of the complete BAC-Col10a1 promoter occurs with high efficiency and specificity in hypertrophic chondrocytes. The BAC-Col10-Cre lines should thus provide a valuable tool to get further insight into the role of genes involved in endochondral ossification by allowing their specific deletion in the hypertrophic zone of the growth plate.

Fig. 1

(a) Diagram showing the position of the Cre recombinase in the BAC-Col10-Cre-frtNeofrt DNA. For the insertion of Cre, the lacZ cassette of the pLacH-Col10a1-LacZ-frtNeofrt vector (Gebhard et al., 2007) was replaced by Cre, and the resulting vector was recombined with BAC-Col10a1 (RP23.192A7) by homologous recombination in E. coli. Correct insertion into exon 2 of Col10a1 was controlled by PCR using primers P1 (Col10a1-Int1) and P5 (“cre reverse”). PCR using primers P1 and P4 served as wt control for endogenous Col10a1. (b) Pulse field gel electrophoresis of BAC Col10a1-LacZ-frtNeofrt DNA after linearization and molecular sieve chromatography on Sepharose 4B. 1) Pulse field markers 10–250 kb; 2)–6) fractions 3–7 of the Sepharose chromatography. Fractions 4 and 5 contain the majority of the BAC DNA which was used for microinjection. (c) Detection of BAC-Col10a1-Cre transgenic offspring by PCR using Primers P1 and P5; 1) markers 2) founder #1466 3) wt 4) Founder #1427; 5) BAC- Col10-Cre DNA.

Fig. 2

X-gal staining for β-galactosidase activity in BAC Col10a1-Cre;ROSA26 embryos and newborns demonstrates strong and specific expression of Cre in all hypertrophic cartilage zones. a–e: newborn (P1), founder line 1427; f–g: E15.5 , founder line 1465. (a) fore limb; (b) ulna and radius, (c) ribs, (d) vertebrae, (e) hindlimbs; f) whole embryo E15.5, g) ribs and fore limb, h) hand , ulna, radius.

Fig. 3

Comparison of β-galactosidase activity in the scapulae of BAC-Col10a1-Cre; ROSA26 transgenic line #1427 (a) , and of the BAC Col10a1 LacZ transgenic line #1520 (b) (Gebhard et al., 2007).

Fig. 4

LacZ is expressed in all hypertrophic chondrocytes in growth plates of BAC-Col10a1-Cre;ROSA26 transgenic mice. a–d: Founder line 1427, P1; e–g. Founder line 1466, E16.5. h,i) founder line 1465, E 15.5. a) fibula; b,g) rib; c) ulna; d,e,i) humerus; h) radius. Paraffin sections, X-gal staining, counterstaining with Eosin. Scale bars: 100 μm.

Fig. 5

Demonstration of hypertrophic chondrocytes in subchondral bone trabeculae escaping resorption and apoptosis. Arrows indicate Col10a1 positive cells located below the resorption front. In situ hybridization with a Col10a1 probe. (a) tibia, (b) humerus of E18.5 wt embryos.