Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells.
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.
Belt
P B
PB
Department of Molecular Genetics, Gorlaeus Laboratories, Leiden, University, The Netherlands.
Groeneveld
H
H
Teubel
W J
WJ
van de Putte
P
P
Backendorf
C
C
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
D000956:Antigens, Viral; D004279:DNA, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; D012313:RNA; D004247:DNA