Development of a cellular model to study alternative splicing of type II collagen gene.
To study regulation of alternative splicing of type II collagen (COL2) pre-mRNA, we constructed a mouse COL2 "minigene" containing genomic sequences spanning exon 1 to exon 4 of COL2 downstream of a cytomegalovirus (CMV) promoter. This minigene was introduced into ATDC5 cells, which undergo chondrocytic differentiation when treated with insulin. Alternative splicing of the COL2 minigene was evaluated by comparing the expression of the two mRNAs transcribed from the minigene to the expression of alternatively spliced transcripts from the endogenous COL2 gene. This analysis suggested that regulation of alternative splicing of pre-mRNAs from the minigene and the endogenous COL2 gene are accomplished by similar mechanisms. We conclude that the cloned genomic fragment contains key sequences necessary for alternative splicing of COL2 pre-mRNA. This system provides a useful model to begin the process of identifying cis- and trans-acting factors that carry out alternative splicing of COL2 pre-mRNA during chondrocyte differentiation.
Hatano
H
H
Department of Orthopedic Research, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Sarkar
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G
Bolander
M E
ME
D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D024043:Collagen Type II / Q000235:genetics*; D003587:Cytomegalovirus / Q000235:genetics; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012322:RNA Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism
D024043:Collagen Type II; D010446:Peptide Fragments; D012322:RNA Precursors; D012333:RNA, Messenger