title: Disruption of the developmentally-regulated Col2a1 pre-mRNA alternative splicing switch in a transgenic knock-in mouse model.
abstract: The present study describes the generation of a knock-in mouse model to address the role of type II procollagen (Col2a1) alternative splicing in skeletal development and maintenance. Alternative splicing of Col2a1 precursor mRNA is a developmentally-regulated event that only occurs in chondrogenic tissue. Normally, chondroprogenitor cells synthesize predominantly exon 2-containing mRNA isoforms (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. Another isoform, IIC, has also been identified that contains a truncated exon 2 and is not translated into protein. The biological significance of this IIA/IID to IIB splicing switch is not known. Utilizing a splice site targeting knock-in approach, a 4 nucleotide mutation was created to convert the 5' splice site of Col2a1 exon 2 from a weak, non-consensus sequence to a strong, consensus splice site. This resulted in apparent expression of only the IIA mRNA isoform, as confirmed in vitro by splicing of a type II procollagen mini-gene containing the 5' splice site mutation. To test the splice site targeting approach in vivo, homozygote mice engineered to retain IIA exon 2 (Col2a1(+ex2)) were generated. Chondrocytes from hindlimb epiphyseal cartilage of homozygote mice were shown to express only IIA mRNA and protein at all pre- and post-natal developmental stages analyzed (E12.5, E16.5, P0, P3, P7, P14, P28 and P70). As expected, type IIB procollagen was the major isoform produced in wild type cartilage at all post-natal time points. Col2a1(+ex2) homozygote mice are viable, appear healthy and display no overt phenotype to date. However, research is currently underway to investigate the biological consequence of persistent expression of the exon 2-encoded conserved cysteine-rich domain in post-natal skeletal tissues.
authors.affiliation: Department of Neurology, Washington University School of Medicine, St Louis, MO 63110, United States.
authors.forename: Renate
authors.initials: R
authors.lastname: Lewis
authors.forename: Soumya
authors.initials: S
authors.lastname: Ravindran
authors.forename: Louisa
authors.initials: L
authors.lastname: Wirthlin
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Traeger
authors.forename: Russell J
authors.initials: RJ
authors.lastname: Fernandes
authors.forename: Audrey
authors.initials: A
authors.lastname: McAlinden
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation; D002678:Chimera; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D047108:Embryonic Development; D005091:Exons; D005260:Female; D055879:Gene Knock-In Techniques; D057809:HEK293 Cells; D006720:Homozygote; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009154:Mutation; D059368:RNA Isoforms / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: C499177:COL2A1 protein, human; C447413:Col2a1 protein, mouse; D024043:Collagen Type II; D059368:RNA Isoforms; D012322:RNA Precursors; D022821:RNA Splice Sites; D012333:RNA, Messenger