title: Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells.
abstract: A cDNA expression vector containing the element oriP and the sequence encoding the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) as well as the hygromycin B-resistance dominant marker gene has been constructed. Its characteristics have been compared to a similar vector lacking the EBV sequences. (a) The EBV+ vector is maintained as an episome with a copy number of approx. 50 per cell, whereas the number of the integrated EBV- copies is in general smaller than 10, when simian virus 40-transformed xeroderma pigmentosum fibroblasts (XP20S-SV) constitute the recipient cell line. (b) The presence of the EBV sequences in the vector resulted in a five- to ten-fold higher transfection efficiency with the Ca.phosphate precipitation technique. (c) cDNA inserts in the EBV+ vector are shown to be efficiently and properly expressed in the recipient cell. (d) If transfection is performed with a mixture of EBV+ vectors with different inserts, transfectants are shown to harbour different plasmids within one cell. (e) The ratio between these plasmids in one cell can be shifted in favour of a vector with a particular insert, when selection for this insert is performed. (f) Reconstruction experiments indicated that isolation of a low-abundance sequence from a mixture of vectors is at least 100-fold more efficient with the EBV+ system, than with the EBV- system. (g) Rescue of the episomal vector from transfected cells can be readily achieved.
authors: Department of Molecular Genetics, Gorlaeus Laboratories, Leiden, University, The Netherlands.
authors: P B
authors: PB
authors: Belt
authors: H
authors: H
authors: Groeneveld
authors: W J
authors: WJ
authors: Teubel
authors: P
authors: P
authors: van de Putte
authors: C
authors: C
authors: Backendorf
mesh_terms: D000956:Antigens, Viral / Q000235:genetics; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D019309:Epstein-Barr Virus Nuclear Antigens; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D012313:RNA / Q000302:isolation & purification; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000956:Antigens, Viral; D004279:DNA, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; D012313:RNA; D004247:DNA