Cloning and characterization of the murinecol11a2 gene, itscDNA, and its functional analysis in transgenic mice

Philipp Vandenberg, Thomas Jefferson University

Abstract

To begin to investigate the role of the cartilage-specific expressed alpha-2 (XI) chain, cosmid clones were isolated that contain the entire murine col11a2 gene. The analysis of these genomic clones led to the determination of a restriction map of the col11a2 locus covering about 30 kb. Overlapping mouse cDNA clones for the col11a2 gene were prepared that cover the entire coding sequence together with the 5$\sp\prime$ untranslated region. Nucleotide sequences of 12.4 kb of contiguous genomic sequence from the 5$\sp\prime$-end of the gene and 4.8 kb of the cDNAs were determined. Analysis of the cDNAs and the transcripts, together with comparison with the genomic sequences was performed, and provided the most complete coding sequences from any species. It revealed the organization of the promoter region, the exon/intron structure for parts of the gene, and the presence of alternative splicing for an exon that codes for a highly acidic domain in the globular amino-terminal propeptide. The chromosomal location and the gene order, and the unusually close proximity of a gene coding for a nuclear receptor for 9-cis retinoic acid were also established. Sequence information from the carboxy-terminal globular domain was utilized to develop a polyclonal antibody specific for the mouse alpha-2 (XI) chain. To gain an understanding of the functional role of the alpha-2 (XI) chain in the homeostasis of cartilaginous tissues in vivo, constructs were prepared that aimed to perturb its physiologic function in transgenic mice. In the first line of experiments, a minigene was constructed that was designed to code for an alpha-chain that would exert a dominant negative effect. Mouse lines carrying the minigene-version of the col11a2 gene were produced, and displayed a mild phenotype resembling human short-limb chondrodysplasias with the additional development of cleft palate in a number of animals. In the second line of experiments, one allele of the col11a2 gene was disrupted in mouse embryonic stem cells by the targeted insertion of a neomycin resistance gene, leading to a deletion of the gene product. Mouse germline chimeras were produced and bred so that offspring lacked one or both alleles of the col11a2 gene. These knock-out animals exhibited a phenotype similar to the minigene animals. Taken together, the results demonstrate a close relationship between fibrillar collagens and alpha-2 (XI) collagen, but also show marked differences in its gene structure and the amino-terminal domain of the protein. The close relationship to the adjacent gene for retinoid X receptor beta raises the question of coordinated expression. Alternative splicing of an exon coding for a highly charged protein domain in neonatal cartilage may be important in development. In transgenic mice, mutations, or the lack of the col11a2 gene product lead to a phenotype closely resembling human chondrodysplasias.

Subject Area

Molecular biology|Cellular biology

Recommended Citation

Vandenberg, Philipp, "Cloning and characterization of the murinecol11a2 gene, itscDNA, and its functional analysis in transgenic mice" (1995). ETD Collection for Thomas Jefferson University. AAI9535025.
https://jdc.jefferson.edu/dissertations/AAI9535025

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