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 ori-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.
Shen
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DuPont Merck Pharmaceutical Co., Experimental Station, Wilmington, DE 19880-0400, USA.
Cooke
G M
GM
Horlick
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RA
D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000235:genetics*; D011945:Receptors, Angiotensin / Q000096:biosynthesis* / Q000235:genetics; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D011945:Receptors, Angiotensin; D001616:beta-Galactosidase