tailieunhanh - Báo cáo khoa học: Fast set-up of doxycycline-inducible protein expression in human cell lines with a single plasmid based on Epstein– Barr virus replication and the simple tetracycline repressor

We have developed a novel plasmid vector, pEBTetD, for full establish-ment of doxycycline-inducible protein expression by just a single transfec-tion. pEBTetD contains an Epstein–Barr virus origin of replication for stable and efficient episomal propagation in human cell lines, a cassette for continuous expression of the simple tetracycline repressor, and a cytomega-lovirus-type 2 tetracycline operator (tetO2)-tetO2 promoter. | ỊFEBS Journal Fast set-up of doxycycline-inducible protein expression in human cell lines with a single plasmid based on Epstein-Barr virus replication and the simple tetracycline repressor Markus Bach1 Silke Grigat1 Barbara Pawlik1 Christian Fork1 Olaf Utermohlen2 Sonia Pal1 David Banczyk1 Andreas Lazar1 Edgar Schomig1 3 and Dirk Grundemann1 3 1 Department of Pharmacology University of Cologne Germany 2 Institute for MedicalMicrobiology Immunology and Hygiene University of Cologne Germany 3 Center for Molecular Medicine University of Cologne CMMC Germany Keywords doxycycline Epstein-Barr virus polyadenylation regulated protein expression tetracycline repressor Correspondence D. Grundemann Department of Pharmacology University of Cologne Gleueler StraBe 24 50931 Cologne Germany Fax 49 221 478 5022 Tel 49 221 478 7455 E-mail Received 17 October 2006 revised 5 December 2006 accepted 5 December 2006 doi We have developed a novel plasmid vector pEBTetD for full establishment of doxycycline-inducible protein expression by just a single transfection. pEBTetD contains an Epstein-Barr virus origin of replication for stable and efficient episomal propagation in human cell lines a cassette for continuous expression of the simple tetracycline repressor and a cytomegalovirus-type 2 tetracycline operator tetO2 -tetO2 promoter. As there is no integration of vector into the genome clonal isolation of transfected cells is not necessary. Cells are thus ready for use 1 week after transfection this contrasts with 3-12 weeks for other systems. Adequate regulation of protein expression was accomplished by abrogation of mRNA polyadenylation. In northern analysis of seven cDNAs coding for transport proteins pools of transfected human embryonic kidney 293 cells showed on off mRNA ratios in the order of 100 1. Cell pools were also analyzed for regulation of protein function. With two transport proteins of the plasma membrane .