Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells
| Autor: | W. J. Teubel, P van de Putte, Peter B.G.M. Belt, Claude Backendorf, H. Groeneveld |
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| Rok vydání: | 1989 |
| Předmět: |
DNA Replication
Herpesvirus 4 Human Genetic Vectors Gene Expression Simian virus 40 Biology medicine.disease_cause Transfection Marker gene Insert (molecular biology) Virus Plasmid hemic and lymphatic diseases Complementary DNA Genetics medicine Escherichia coli Tumor Cells Cultured Humans Vector (molecular biology) Cloning Molecular Antigens Viral Cell Line Transformed General Medicine DNA Epstein–Barr virus Virology Molecular biology Epstein-Barr Virus Nuclear Antigens DNA Viral RNA HeLa Cells Plasmids |
| Zdroj: | Gene. 84(2) |
| ISSN: | 0378-1119 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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