So far, more than 150 genetic loci associated with bone mineral density have been discovered by genome-wide association studies (GWAS). These genetic loci represent potential biomarkers for early detection of osteoporosis or potential new therapeutic targets for osteoporosis. One of the newly discovered loci, rs7851693, is a single nucleotide polymorphism in the intron of the gene for FUBP3 (“Far upstream binding protein 3”). The roles of FUBP3 in bone metabolism are not yet fully understood. To establish a groundwork for functional genomics experiments, which could potentially help to determine the role of FUBP3 dysregulation, we prepared two plasmid constructs. We used Gibson cloning to assemble a plasmid for FUBP3 overexpression (pFLAG-CMV-1-FUBP3) and FUBP3 knockout by CRISPR/Cas9 (pX459-gRNA1-FUBP3). Two cell lines were transfected with the plasmids – HEK 293T, derived from human embryonic kidney cells, and A549, derived from lung cancer cells. Western blotting was used to confirm the changes in FUBP3 expression in the HEK 293T cell line after transfection. Sequencing was used to determine the nucleotide sequence of the gene in the HEK 293T cell line, which was subject to change after genome editing with the CRISPR/Cas9 system. Finally, indirect immunofluorescence was used to show the localization of the FUBP3 protein in the cell. In the A549 cell line, FUBP3 is localized predominantly in the cytoplasm. The two assembled plasmid constructs are a basis for further functional assays that will potentially help us understand the role of FUBP3 and its dysregulation.
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