| Popis: |
To study alternative glycosylation of immunoglobulin G (IgG), which is functionally important in pro- or anti-inflammatory effector function of the antibody, we developed a system for IgG production in the model cell line HEK-293F, which we modified by incorporating the CRISPR/dCas9-based molecular machinery for epigenetic regulation of gene expression. We extended our existing modular system for CRISPR/dCas9 fusions facilitating gene regulation to enable its stable integration into HEK-293F cells, thus creating derived cell lines with integrated machinery for programmed transcriptional control. A key factor for targeting the dCas9 fusions with activators and repressors, the guide RNA (gRNA) was transiently transfected into the derived cell lines on a separate plasmid, that also contains a cassette for monocistronic expression of IgG heavy and light chain. In this bipartite system, the creation of derived cell lines with CRISPR/dCas9 components eliminates the inefficiency of transfection with a large construct, while combining a key element for targeting (gRNA) with the IgG expression cassette on a small plasmid facilitates high transient transfection efficiency while virtually eliminating the background from untransfected cells. We validated the system by up- or downregulating the known glycosyltransferases. Transcriptional up- and downregulation of B4GALT1, responsible for adding galactose to the glycan core, had the expected effect on the abundance of agalactosylated and galactosylated structures, changing the glycosylation profile according to the transcript level. Downregulation of FUT8, encoding a fucosyltransferase, decreased the core-fucosylated structures, while upregulation of ST6GAL1 and MGAT3 increased sialylated structures and those with a bisecting GlcNAc, respectively, in line with our expectations. The system is now being used to further study the role of genes associated with IgG glycosylation in GWA studies. It can also be easily repurposed to serve as a model for other proteins and their posttranslational modifications, with appropriate targeting via gRNA. Finally, to eliminate the frequently raised concern about suitability of the HEK293-F system as a model for plasma cells, we are currently adapting the system for use with lymphoblastoid cell lines (LCLs). |
