Characterization of FAM65B knock-out mice and role of FAM65B in skeletal muscle stem cells differentiation
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The family with sequence similarity 65, member B (Fam65b) protein is thought to facilitate fusion of myocytes and formation of myotubes during the differentiation of human myogenic cells. Fam65b and histone deacetylase 6 (HDAC6) co-immunoprecipitate and together regulate the levels of acetylated tubulin, which might control microtubule stability in myogenic cells. In this thesis, to gain further insight on the role of Fam65b in the differentiation pathway and motility of myogenic cells, we characterized a Fam65b knock-out (KO) mouse model. Genotyping and transcriptional analysis revealed that a thirteen exons-long region of the Fam65b gene has been successfully ablated and the mRNA amplicons within the deleted segment are not transcribed. Nevertheless, mRNA products corresponding to genomic regions downstream of the deleted area are still detected. Furthermore, analysis of skeletal muscle lysates via western blot (WB) does not show a complete loss of Fam65b expression, but only reduced translation of some isoforms. Nevertheless, WBs of myogenic cells that have been directly isolated from Fam65b KO mice and expanded in vitro revealed the absence of a 120 Kd band, which putatively corresponds to the long isoform of Fam65b. Finally, our data show that Fam65b KO mice are significantly heavier than wild type (WT) mice, and that this phenotype is consistently observed across both genders during the first seven months of age. While functional and molecular analyses of the KO mouse model are still ongoing, future work might include generating a new KO model via the CRISPR-Cas9 technology to ablate all isoforms of Fam65b.