Involvement of phosphoinositide 3-kinase signaling pathway in chondrocytic differentiation of ATDC5 cells: application of a gene-trap mutagenesis

Journal of Cellular Biochemistry
Miyuki Ihara-WatanabeToshimi Michigami

Abstract

Gene-trap mutagenesis is based on the notion that the random insertion of a trapping vector may disturb the function of inserted genes. Here, we applied this method to murine mesenchymal ATDC5 cells, which differentiate into mature chondrocytes in the presence of insulin. As the trap vector we used pPT1-geo, which lacks its own promoter and enhancer, but contains a lacZ-neo fusion gene as a reporter and selection marker driven by the promoter of the trapped gene. After pPT1-geo was introduced into ATDC5 cells by electroporation, the neomycin-resistant clones were screened for beta-galactosidase activity. The selected clones were cultured in differentiation medium to evaluate the chondrogenic phenotype. The clones no. 6-30 and 6-175, which exhibited impaired and accelerated mineralization, respectively, were subjected to further analysis. In clone no. 6-30 in which the gene coding for the p85alpha subunit of phosphoinositide 3-kinase (PI3K) was trapped, the expression of marker genes of early chondrocytes including collagen type II, aggrecan, and PTH/PTHrP receptor was delayed. The insulin-induced stimulation of growth was reduced in clone no. 6-30 compared with the parental ATDC5 cells. Moreover, treatment of parental ATDC5 cel...Continue Reading

References

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Citations

Mar 15, 2012·The Journal of Biological Chemistry·Takao KoshimizuToshimi Michigami
Mar 15, 2008·Biochemical and Biophysical Research Communications·Hiroaki WatanabeB Mark Evers
Dec 4, 2009·The Anatomical Record : Advances in Integrative Anatomy and Evolutionary Biology·Erik Kern HarringtonKathy K H Svoboda
Aug 18, 2020·Biomedical Research·Wataru AriyoshiTatsuji Nishihara

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