Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells
1989; Elsevier BV; Volume: 84; Issue: 2 Linguagem: Inglês
10.1016/0378-1119(89)90515-5
ISSN1879-0038
AutoresPeter B.G.M. Belt, Herman Groeneveld, Wilma J. Teubel, Pieter van de Putte, Claude Backendorf,
Tópico(s)Herpesvirus Infections and Treatments
ResumoA 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.
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