Development of GFP-Tagged Akata EBV Cell Line via BACmid System
The overall aim of this project is to generate a GFP-tagged Akata EBV-infected immortalized B cell line using the BACmid system. Epstein-Barr virus (EBV) is a gammaherpesvirus that has infected over 90% of adults globally, and it is associated with a variety of different conditions, including infectious mononucleosis (IM) in acute infection and nasopharyngeal cancers (NPC), Burkitt's Lymphoma (BL), gastric cancers (GC), and certain autoimmune conditions during latency. Within the body, EBV can exist in two phases: the lytic phase, which is a phase of active replication, and the latent phase, in which viral DNA existing in episomes undergoes limited transcriptional and translational action. The virus can undergo lytic replication in epithelial and B cells and can establish latency in B cells. There are several different strains of the virus, including Akata EBV, which is associated with a tumor cell line established from an individual with BL. In order to study the mechanics of cell-to-cell infection, green fluorescent protein (GFP) can be used as a tag in order to detect individually infected cells using fluorescent microscopy.
The project involved replication of the plasmid (gifted to the Robertson Lab by the Lin Lab of the Tulane University School of Medicine) in E. coli, collection of the plasmid in solution, transfection of HEK-293 cells, induction of the lytic phase in the HEK-293 cells, collection of the viral supernatant, and infection of primary B cells. Replication of the E. coli was conducted in LB with kanamycin (due to the specific prokaryotic antibiotic resistance gene found on the gifted plasmid). Once replicated, the plasmid was harvested using QIAGEN maxipreparations, and the harvested plasmid was used to transfect HEK-293 cells with jetPRIME transfection reagent. The lytic phase was induced in the transfected cells, and the resulting viral supernatant was used to infect PBMCs.
Though the project was not seen to completion, the successful induction of the lytic phase and collection of viral supernatant is extremely promising for the prospects of this project. The resulting GFP-tagged Akata-producing cell line will hopefully contribute to ongoing research that can help determine the dynamics of Akata EBV infection and eventually prevent or treat the conditions associated with Epstein-Barr virus.