Genetic Alteration of Selection Resistance on BAC-EBV Plasmids by Homologous Recombination and Establishment of the Stably Transfected Cell Line
Lielian Zuo, Meijuan Zhu, Shujuan Du, Qiao Li, Guiyuan Li, Jianhong Lu
Available Online November 2015.
- https://doi.org/10.2991/itms-15.2015.254How to use a DOI?
- Epstein-Barr virus; homologous recombination; BAC; GFP
- To obtain recombinant Epstein-Barr virus (EBV) particles with the neomycin resistance from BAC-EBV with hygrymicin (hyg) gene, homologous recombination was employed for altering the original hyg gene to neo resistance gene in the BAC-EBV genome. The kanamycin marker gene (kan) containing the recombinant protein FLP recognition sites (FRTs) was connected to the neo gene in the vector of pGEM-T using conventional cloning techniques. Both ends of the connective are the arms with 20bp of homology sequences responding to the ends next to the hyg gene. PCR and cloning techniques were further performed to extend homology arms to make them reach 89bp. The linear fragment kan-neo with 89bp of arms was then electroporated into the bacteria containing BAC-EBV genome. Under the mediation of the red system, the hyg gene in BAC-EBV was replaced by neo gene. A series of methods including antibiotic selection, PCR amplification, sequencing and restriction enzyme digestion were used for the identification of the mutated plasmids. The results showed that BAC-EBV/neo, was constructed as expected. Through the transfection of the BAC-EBV/neo plasmid, G418 selection, and GFP observation, we established 293-EBV/neo cell line. This research lays a foundation for the establishment of broader cell models in the study of EBV-associated pathogenic mechanisms.
- Open Access
- This is an open access article distributed under the CC BY-NC license.
Cite this article
TY - CONF AU - Lielian Zuo AU - Meijuan Zhu AU - Shujuan Du AU - Qiao Li AU - Guiyuan Li AU - Jianhong Lu PY - 2015/11 DA - 2015/11 TI - Genetic Alteration of Selection Resistance on BAC-EBV Plasmids by Homologous Recombination and Establishment of the Stably Transfected Cell Line PB - Atlantis Press SP - 1051 EP - 1054 SN - 2352-538X UR - https://doi.org/10.2991/itms-15.2015.254 DO - https://doi.org/10.2991/itms-15.2015.254 ID - Zuo2015/11 ER -