Fifth International Conference on the Molecular Biology and Pathology of Matrix 409

2. Type II Collagen

Abnormal Chondrocyte Differentiation and Chondrodysplasia in Transgenic Mice

K.S.E. Cheah*, A.W.K. Wai*, A. Levy*, P.A. Trainort, T. Kuffner* and P.P.L. Tarot

*Biochemistry Department, Hong Kong University, Hong Kong; and tEmbryology Unit, Children's Medical Research Institute, Wentworthville, Australia

Mutations in the human ~1(II) collagen (COL2A1) gene have been implicated in some of the inherited human chondrodysplasias. But a causal relationship between abnormal chondrocyte differentiation and/or proliferation and chondrodysplasia has not been demonstrated. Since the differentiated phenotype of chondrocytes is known to be sensitive to oncogene expression, we have tested the ability of SV40 T anti- gen to cause abnormalities in cartilage development. The cis-regulatory elements of the COL2A1 gene have previously been used to target expression of SV40 T antigen to differentiating chondrocytes in transgenic mice. The phenotypic consequences of transgene (pAL21) expression in transgenic mice is malformed skeleton, disproportionate dwarfism and perina- tal/neonatal death. However, due to the dominant perinatal lethal effect of the Tag transgene, a devel- opmental analysis of the effect of COL2A1 targeted Tag expression on chondrocyte differentiation and on postnatal skeletal development was not possible. To overcome this difficulty, ES cells which carry stably integrated copies of pAL21 were used to produce mouse chimeras. Chimeras have survived to neonatal stages but were smaller than normal non-chimeric mice and displayed severe kyphosis and retarded ossi- fication of the skeleton. Islands of disorganized im- mature chondrocytes wi th decreased amounts of matrix in the epiphyseal growth plate of long bones were found in pAL21 chimeras. These clusters of chondrocytes failed to organize themselves into char- acteristic columns of proliferating cells. (xl(II) colla- gen mRNA was markedly reduced for chondrocytes in these islands compared to their normal counterparts. Other cartilage characteristic mRNAs, such as aggre- can and ~2(XI) collagen, were reduced to a lesser degree for those chondrocytes within these islands compared to properly organized surrounding chon- drocytes. The primary cause of the skeletal abnormality in pAL21 chimeras may be an impaired ability of chondrocytes to differentiate and an associated paucity of cartilage matrix. The COL2A1- Tag transgene in mouse ES cells and chimeras provide tools for analyzing the complex relationships between differen-tiation and phenotype during skeletogenesis and the developmental basis of dominant lethal forms of chondrodysplasia.

Expression of the Human COL2A1 Gene in Transgenic Mice Results in Growth Retardation

K.S.E. Cheah*, K.K.Y. Ho*, J. Lun*, M. Pangt and P.P.L. Tams

*Department of Biochemistry and tOral Biology Unit, The University of Hong Kong, Hong Kong; and :~Embryology Unit, Children's Medical Research Institute, Wentworthville, Australia

Human and mouse 0tl(II) collagens are highly conserved with amino acid sequence homology greater than 90%. To investigate the possibility of interspecies association of 0tl(II) chains and also to study the phenotypic consequences of the synthesis of a mouse-human hybrid type II collagen, we have generated two transgenic mouse lines, numbers 277 and 1085. Both lines carry intact copies of the human COL2A1 gene but with differing lengths of 5'- flanking DNA sequence. All transgenic animals were shown to express the human transgene. Growth retardation was observed in mice from both lines but 277 animals were more severely affected than 1085 mice. In addition, within line 277, the degree of growth retardation was heterogenous being either moderate or severe. The expression levels of both the human COL2A1 transgene and the endogenous mouse type II collagen gene in both transgenic lines were analysed by RNAse protection assays. All line 277 animals expressed a higher level of the COL2A1 gene when compared to line 1085. Most severely growth retarded mice expressed the highest levels of COL2A1 mRNAs. In addition, levels of the endogenous mouse type II collagen mRNA were found to be elevated in all transgenic animals compared to non-transgenic littermates. The mRNA levels of other genes which are also expressed in the extracellular matrix of cartilage such as type IX collagen, aggrecan and link protein were found to be unaltered in these transgenic mice. These data raise interesting questions about the possibility of interspecies associations of collagen 0~ chains and the relationship between levels of type II collagen expression and neonatal growth.