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Sharda P Yadav, Lijin Dong, Anand Swaroop; Mouse embryonic fibroblast derived from Ccp110 knockout mouse leads to aberrant elongation and defective segregation of centrioles and failed to produce cilia. Invest. Ophthalmol. Vis. Sci. 2014;55(13):396.
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© ARVO (1962-2015); The Authors (2016-present)
In most of the mitotic animal cells centrosome duplication occurs at G2-M phase and defect in it accompanied by extensive chromosomal abnormalities. Purpose of this study to understand the mechanism of centrosome biogenesis using mouse embryonic fibroblast derived from Centrosomal coiled coil protein 110 (Ccp110) knockout mouse.
Ccp110 knockout mouse were generated and mouse embryonic fibroblasts (MEFs) were derived from wild type heterozygous and Ccp110 knockout E 12.5 embryos. Cilia were developed by serum starving the cultures cells and looked for different cilia markers. Centrosomal abnormalities were looked by immunohistochemistry with specific centriole markers. FACS analysis of the MEFs was performed for cell cycle analysis after staining of the DNA with propidium iodide.
Staining of the cilia with acetylated tubulin and ARL13b suggested that almost 90 % of the Ccp110 knockout MEFs failed to develop cilia. Ccp110 heterozygous MEFs were able to developed cilia normally as wild type. Suggesting that one copy of the Ccp110 is sufficient for normal development of cilia in MEFs. Analysis of the centrioles by staining with γ-tubulin revealed that a subset of Ccp110 mutant MEFs failed to segregate newly formed centrioles and elongated in size compared to wild type MEFs. Further staining of the centrioles with Ninein showed a weak staining in Ccp110 mutant MEFs compared to wild type cells suggesting normal development of the sub distal appendages. Cell cycle analysis suggested a defect in the G2-M phase with accumulation of approximately 40% of polyploidy (8N) cells in Ccp110 mutant MEFs compared to wild type. Our results suggested a critical role of Ccp110 in regulations of centriole length and cilia formation in MEFs.
We have successfully generated a Ccp110 knockout mouse. Mouse embryonic fibroblast derived from Ccp110 knockout failed to develop cilia, probably due to aberrant segregation of daughter centrioles, which resulted in the accumulation of G2-M phase cells with increased polyploidy. Further analysis of the mutant centrioles with transmission electron microscopy (TEM) is under progress and will allow us to further understand the ultra structural defects in it compared to wild type centrioles.
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