Genetic Alteration Of Selection Resistence On BAC-Based Plasmids By Homologous Recombination And Establishment Of BAC-EBV Infected Cell Lines

EB virus (Epstein-Barr virus, EBV) is associated with kinds of human tumors, such as nasopharyngeal cancer and Burrkitt lymphoma. Due to the large genome with172kb, EBV is not easy for the genetic manipulation during the viral study in the context of the whole genome. In experiments, in order to solve this problem, the E. Coli F factor as used in the bacterial artificial chromosome (BAC) vector was introduced into the genome of EBV. The F factor ensures the genome replication with a low-copy number (1copy) in bacteria strictly. BAC-based EBV genome can accurately replicates with the BAC’s replication, and is possible for the genetic modification in bacteria. On the other hand, in vitro, EBV genome is lost easily in continuous passages from primarily EBV-positive nasopharyngeal carcinoma (NPC) cell lines. Moreover, EBV can not infect epithelial cells due to the lack of the receptor CR2. Therefore, it is difficult to produce EBV-infected epithelial cell models in vitro. This problem hinders the study of the EBV-associated NPC.As previously described, the constructs containing genes BZLF1and BALF4of EBV was co-transfected into the293HEK/p2089cells derived from the BAC-based plasmid, p2089(BAC-EBV), with the presence of hygromycin resistence gene (hyg) for selection in eukaryotic cells. EB virus particles were thus induced. The EB virus particles infect B lymphocytes, and then "transfered" to EBV-negative NPC cells through the method of cell-to-cell contact. The visualization with GFP and the RT-PCR analysis of the genes LMP1and LMP2indicated that we successfully established the method of "transfer infection" for the produce of EBV-infected NPC cell lines in our lab. However, the strong sensitivity of the nasopharyngeal epithelial cells to hygromycin leads to thedifficulty for the the stable selection. Therefore, we designed to alter the original hyg gene to puromycin resistance gene (pac) in the BAC-EBV genome. The kanamycin marker gene (kan) containing the recombinant protein FLP recognition sites (FRTs) was connected to the pac genein the vector of pcDNA3.1(+) using conventional cloning techniques. Both ends of the connective are the arms with20bp of homology sequences responding to the ends next to the hyg gene. PCR and cloning techniques were further employed to extend homology arms to make them reach89bp. The linear fragment kan-pac with89bp of arms was then electroporated in the bacteria containing BAC-EBV genome. With the mediation of the γ phage redαβγ system, the hyg gene in p2089was replaced by pac gene through the homologous recombination (also called ET cloning). The introduced kan selection gene was then specifically removed by the recombinant protein FLP, leaving a FRT "scar" of69bp. By using the same techniques of linear transformation and ET cloning, the hyg gene in the blank plasmid, pM-BAC, was also replaced by the pac geneA series of methods including antibiotic selection, PCR amplification and cloning, sequencing and restriction enzyme digestion were used for the identification of the mutated plasmids. The results showed that the gene alterations, resulting in the plasmids, pBAC-EBV/pac and pM-BAC/pac respectively, were completed as expected. Through the transfection of the pBAC-EBV/pac and pM-BAC/pac plasmids’ and puromycine selection, we established BAC-EBV/pac293epithelial cell lines. This research lays a foundation for the establishment of cell models in the study of EBV-associated nasopharyngeal carcinoma.