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Abstract:
The most complex part of the vertebrate body is the head. This intricate structure forms during early embryonic development. However, it remains unclear which genetic and developmental mechanisms underlie the wide variability in head formation and shaping.Many studies have identified genes that play a fundamental role in head development. One of the genes with a prominent function in skull formation is the de-novo evolved meningioma 1 (Mn1). Genome editing tools such as CRISPR/Cas9 offer excellent opportunities to study gene function. Herein, the aim was to establish a mutant cell line from the murine osteogenic cells MC3T3-E1. Therefore, CRISPR/Cas9 methodology was employedto generate perturbations in the Mn1gene. Whether such genetic perturbation was successful has yet to be validated. Additionally, to carry out the functional validation of the disrupted Mn1gene in the mutant cell line, the in vitromodel of osteoblast differentiation was optimised, together with a battery of staining procedures to assess the levels of alkaline phosphatase and Calcium during this process. The staining procedures were optimisedusing the original unperturbed MC3T3-E1. The resultsshow that the cells can differentiate into osteoblasts, and the staining methods BCIP/NBT and Alizarin Red S reliably detect the differentiation hallmarks. Furthermore, thedifferentiation capacity of these cells depends on their age (~passage), which must be taken into consideration for future experiments.
