transgenic mice were crossed with Rosa26 reporter mice in which expression of Escherichia coli
β-galactosidase can be induced by Cre-mediated recombination6
. Offspring were genotyped by PCR using Cre-specific primers and primers for detecting R26R
alleles. The sequences of PCR primers for genotyping Col2a1-CreERT2
mice are: 5′-CCTGGAAAATGCTTCTGTCCGTTTGCC-3′ (forward primer) and 5′-GAGTTGATAGCTGGCTGGTGGCAGAG-3′ (reverse primer) and the size of the PCR product is 600-bp. The sequences of PCR primers for genotyping Rosa26R reporter mice are: R1295, 5′-GCGAAGAGTTTGTCCTCAACC-3′; R523, 5′-GGAGCGGGAGAAATGGATATG-3′ and R26F2, 5′- AAAGTCGCTCTGAGTTGTTAT-3′. The 600-bp PCR product was detected in wild-type mice and the 325-bp PCR product was detected in homozygous Rosa26R mice. In heterozygous Rosa26R mice, both 600 and 325-bp PCR products were detected.
To determine if the Col2a1-CreERT2 transgenic mice can be used to target genes expressed in articular chondrocytes in adult mice, they were bred with Rosa26 reporter mice to follow the time course of reporter expression. TM induction was performed in 2-week-old Col2a1-CreERT2;R26R transgenics (1 mg TM/mouse/day for 5 days) and mice were sacrificed 1 and 6 months after the last injection. The long bones were harvested and fixed in 0.2% glutaraldehyde, decalcified, and processed for frozen sectioning followed by X-Gal staining. Nuclear Fast Red staining was performed as a counter stain. The recombination efficiency was determined by counting the X-Gal-positive cells divided by the total number of cells on the articular surface (n=4). The results showed that TM induced efficient Cre-recombination in articular chondrocytes 1 and 6 months after TM induction (). Eighty five percent and 82% Cre-recombination efficiency was achieved in articular cartilage area of these mice respectively () (n=4). It should be noted that some cells in the deep zone of the articular cartilage were negative for X-Gal staining; this may be due to the poor penetration of tamoxifen into these areas.
Fig. 1 Histological analyses of the Col2a1-Cre+/-ERT2;R26R+/- mice. 2-week-old Col2a1-Cre+/-ERT2;R26R+/- double transgenic mice and Cre-negative control littermates were injected with TM (1 mg/mouse/day for 5 days). Mice were sacrificed 1 (a) and 6 months ( (more ...)
Overall, these results suggest that Col2a1-CreERT2 transgenic mice provide a strategy for causing tissue specific gene deletion in a temporal manner. Since the percentage of X-Gal-positive articular chondrocytes is similar at 1 and 6 months after TM induction, there is likely minimal turnover or cell renewal in articular cartilage, consistent with the limited reparative potential of this tissue.
Using the 3 kb Col2a1 promoter, Ovchinnikov et al.8
transgenic mice which show efficient Cre-recombination in chondrocytes during mouse development. A similar approach was also reported by Nakamura et al.9
who generated Col2a1-CreERT
transgenic mice using the same Col2a1 promoter construct as we used in our transgenic mice. Although efficient Cre-recombination was demonstrated in the embryonic and postnatal skeleton, it becomes undetectable in 12-week-old Col2a1-CreERT
. Several factors such as differences in transgene constructs, integration sites and copy numbers may contribute to these phenotypic distinctions.