GENERATION OF A NOVEL RECOMBINANT MOUSE MODEL EXPRESSING ONLY ONE COL2A1 ISOFORM
Audrey Mcalinden
Orthopaedic Surgerywashington University
Grant 5R21AR053513-02 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases, IRG: SBDD
Abstract: 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
Project start date: 2007-09-01
Project end date: 2009-08-31
Sponsored Links Lab Supply Mall http://www.labsupplymall.com
Grants awarded to Audrey Mcalinden
GENERATION OF A NOVEL RECOMBINANT MOUSE MODEL EXPRESSING ONLY ONE COL2A1 ISOFORM
Audrey Mcalinden
Orthopaedic Surgerywashington University
Grant 5R21AR053513-02 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases, IRG: SBDD
Abstract: 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
Project start date: 2007-09-01
Project end date: 2009-08-31
Related Publications
Upregulation of Runx2 and Osterix during in vitro chondrogenesis of human adipose-derived stromal cells. Biochem Biophys Res Commun. 2008 Jul 18; 372( 1): 230-5. Epub 2008 May 13. PMID: 18482578 A novel tumor necrosis factor alpha-responsive CCAAT/enhancer binding protein site regulates expression of the cartilage-derived retinoic acid-sensitive protein gene in cartilage. Arthritis Rheum. 2008 May; 58( 5): 1366-76. PMID: 18438857 Expression of two novel alternatively spliced COL2A1 isoforms during chondrocyte differentiation. Matrix Biol. 2008 Apr; 27( 3): 254-66. Epub 2007 Oct 18. PMID: 18023161 Missense and nonsense mutations in the alternatively-spliced exon 2 of COL2A1 cause the ocular variant of Stickler syndrome. Hum Mutat. 2008 Jan; 29( 1): 83-90. PMID: 17721977 Mechanisms of disease: role of chondrocytes in the pathogenesis of osteoarthritis--structure, chaos and senescence. Nat Clin Pract Rheumatol. 2007 Jul; 3( 7): 391-9. PMID: 17599073 
Nuclear protein TIA-1 regulates COL2A1 alternative splicing and interacts with precursor mRNA and genomic DNA. J Biol Chem. 2007 Aug 17; 282( 33): 24444-54. Epub 2007 Jun 19. PMID: 17580305 Alternative splicing of type II procollagen exon 2 is regulated by the combination of a weak 5' splice site and an adjacent intronic stem-loop cis element. J Biol Chem. 2005 Sep 23; 280( 38): 32700-11. Epub 2005 Aug 2. PMID: 16076844 Quantification of mRNA expression levels in articular chondrocytes with PCR technologies. Methods Mol Med. 2004; 100: 79-100. PMID: 15280589 Regulation of protein diversity by alternative pre-mRNA splicing with specific focus on chondrogenesis. Birth Defects Res C Embryo Today. 2004 Mar; 72( 1): 51-68. Review. PMID: 15054904 Alpha-helical coiled-coil oligomerization domains are almost ubiquitous in the collagen superfamily. J Biol Chem. 2003 Oct 24; 278( 43): 42200-7. Epub 2003 Aug 14. PMID: 12920133 Trimerization of the amino propeptide of type IIA procollagen using a 14-amino acid sequence derived from the coiled-coil neck domain of surfactant protein D. J Biol Chem. 2002 Oct 25; 277( 43): 41274-81. Epub 2002 Aug 22. PMID: 12194968 Processing of type II procollagen amino propeptide by matrix metalloproteinases. J Biol Chem. 2002 Jan 18; 277( 3): 2193-201. Epub 2001 Nov 8. PMID: 11705992