GENERATION OF A NOVEL RECOMBINANT MOUSE MODEL EXPRESSING ONLY ONE COL2A1 ISOFORM

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DESCRIPTION (provided by applicant): The present proposal describes a novel knock-in strategy to generate a recombinant mouse expressing the type IIA procollagen isoform only. The novel aspect of this approach involves altering the splice site of the regulated exon in the type II procollagen gene (Col2a1) such that the exon will always be included in the final mRNA. In doing so, the natural cellular splicing mechanisms will be utilized which is important given the increasing reports that mechanisms of transcription and pre-mRNA splicing are tightly co-ordinated in the nucleus. Our model represents an important alternative to other recombinant "knock-in" strategies where intron-less cDNAs are commonly introduced into the genomic area of interest. If successful, application of our proposed knock-in technique can be used to manipulate splice site sequences of a regulated exon within any gene of interest. Therefore, invaluable information on the in vivo biological function of different protein isoforms derived from the same gene will be gained. This is particularly appealing in this post-genome sequencing era where we now know that the majority of protein diversity results from alternative splicing of pre-mRNA. Alternative splicing of Col2a1 is developmentally-regulated where exon 2 is spliced (included) by chondroprogenitor cells forming type IIA procollagen while differentiated chondrocytes exclude exon 2 forming type IIB procollagen. As this IIA-IIB switch is specific to cartilage development, we hypothesize that alternative splicing of Col2a1 is an essential commitment mechanism required for correct development of cartilage. We propose to alter four intronic nucleotides at the 5' splice site of exon 2 that we have shown, by in vitro methods utilizing a COL2A1 mini-gene, will result in constitutive splicing of exon 2, thereby only producing the type IIA procollagen isoform. We decided to express type IIA in mice to potentially avoid lethality problems since this isoform is also expressed early during development of other tissues such as heart and kidney. If successful, we predict that the IIA mouse will contain abnormal cartilage due to a deficiency in the "mature" cartilage type IIB collagen fibers that normally make up the majority of the extracellular matrix. Subsequently, long bone formation may also be affected. This novel model system will provide significant insight into the biological function of the Col2a1 isoforms during chondrogenesis and also provide additional knowledge related to cartilage repair mechanisms since the type IIA procollagen isoform has also been shown to be re-expressed during osteoarthritis.