IMPROVED EXPRESSION CLONING USING REPORTER GENES AND EPSTEIN-BARR-VIRUS ORI-CONTAINING VECTORS

Levels of expression of two reporter genes cloned into SV40 or Epstein-Barr virus (EBV) ori-containing plasmids were measured following transient transfection of cell lines constitutively expressing T-antigen or EBV nuclear antigen 1 (EBNA1). The TSA201 and COS7 cell lines stably produce T-antigen and support replication of the SV40 ori-containing constructs while the 293EBNA cell line produces EBNA1 and supports replication of EBV osi-containing plasmids. We found that 293EBNA cells express >25-fold more beta-galactosidase (beta Gal) per mg protein than COS7 cells and 11-fold more beta Gal than TSA201 cells. We also demonstrate that 293EBNA cells are able to express 70-100-fold more angiotensin II type-1 receptor (AT1) per mg protein than COS7 or TSA201 cells. We examined the suitability of each cell line for use in expression cloning using a NaOH 'scrape' method as an improvement over emulsion autoradiography for detection, Measurable AT1 signals can be detected when reporter plasmids are diluted up to 1000-fold for COS7 and TSA201 cells, and up to 80 000-fold for 293EBNA cells. These data demonstrate that 293EBNA cells offer a significant improvement in expression cloning technology as compared to the conventionally used T-antigen-based cell lines.