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P&F Grant Awards

Year 2


Grant # 3

Generation of a knock-in mouse model to study the role of type II collagen alternative splicing during chondrogenesis

PI: Audrey McAlinden, Ph.D.,

 

Specific Aims

Generation and analysis of recombinant knock‐in mice expressing only one isoform of type II collagen.

Rationale: Alternative splicing of the type II collagen gene (Col2a1) occurs in a developmentally‐regulated manner and only in chondrogenic tissues. Chondroprogenitor cells synthesize Col2a1 mRNA isoforms containing exon 2 while differentiated chondrocytes generate Col2a1 isoforms devoid of exon 2. The significance of this alternative splicing switch in skeletal development is not known.

Hypothesis: We hypothesize that Col2a1 alternative splicing is necessary to generate proper cartilage tissue which subsequently results in normal endochondral bone formation.

Approach: We plan to inhibit the Col2a1 developmentally‐regulated splicing switch in vivo by generating knock‐in mice that will only synthesize type IIA collagen (the exon 2‐containing isoform normally produced by chondroprogenitor cells) while production of type IIB collagen (the isoform devoid of exon 2 normally produced by differentiated chondrocytes) will be completely inhibited. This mouse model will directly address the role of Col2a1 alternative splicing in chondrogenesis and will aid in defining functional roles of these distinct protein isoforms. Recently, we have identified and confirmed positive ES clones that will be used for blastocyst injections. Therefore, in this one year Pilot & Feasibility Study Proposal, we aim to generate the knock‐in animals, carry out preliminary analyses of embryos and post‐natal mice and develop an in vitro based ES cell differentiation assay system. Services provided by the Mouse Genetics Models Core D and the in situ Molecular Analysis Core C (affiliated with the Center of Musculoskeletal Research) will be used extensively. We predict the knock‐in mouse model will provide new and important insights into the mechanisms involved in regulating cartilage and bone development. This proposal therefore provides a unique opportunity to permit the progress of this currently unfunded research project and to generate strong preliminary data for a future RO1 application.