An EBV-based mammalian cell expression vector for efficient expression of cloned coding sequences

Comparison of mammalian cell expression vectors with and without an EBV-replicon

“…Recombinant plasmids CMV-cat, EBV-CMV-cat (also designated pKTH 536 and pKTH 540) [10], pSV 2-cat (obtained from B. Howard, NIH, Washington, DC, U.S.A.), and Py 3 (obtained from K. Blochlinger, Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland) [4], were propagated in Escherichia coli B10. M13mpl8 and M 13rap 19 were propagated in E. coli JM 103.…”

“…of Ziirich, Switzerland), and 293 cells (transformed human embryonal kidney, from U. Pettersson, Univ. of Uppsala, Sweden) were cultured and transfected as described previously [10]. The transfected ceil clones were selected with 300 gg/ml of Hygromycin B and after two weeks the resistant cell clones were isolated from the master plates by scratching.…”

“…The detector probe reagent for sandwich hybridization was obtained by religating the remaining 3.4 kb Eco RI fragment of CMV-cat. The EBV-CMV-cat copy numbers were determined as described earlier [10].…”

“…We have recently described an efficient mammalian cell expression vector [10] consisting of Epstein-Barr virus DNA fragments oriP and EBNA-1 for extrachromosomal replication [13,[21][22][23], and of a mammalian expression cassette containing the cytomegalovirus (CMV) immediate early gene enhancer [3,5,7] coupled with the SV40 early promoter and transcription processing signals. The vector replicates extrachromosomally in h u m a n and m o n k e y cell lines with up to 20 copies per cell.…”

“…The vector replicates extrachromosomally in h u m a n and m o n k e y cell lines with up to 20 copies per cell. High levels of expression of the bacterial chloramphenicol acetyltransferase (CAT) gene was obtained [10].…”