Evaluation of the T-REx transcription switch for conditional expression and regulation of HSV-1 vectors.
Herpes simplex virus 1 (HSV-1) strain ANG and ANGpath were cloned as bacterial artificial chromosome (BAC). Two different types of BAC genomes were obtained. BAC genomes of type I contained the BAC replicon at the intended target region between the genes of UL48 and UL49. In BAC genomes of type II, the BAC sequences were found to be aberrantly fused between the termini of the HSV-1 genome. Both the BAC types were used to establish a conditional gene expression system for HSV-1 by Flp recombinase-mediated insertion of expression vectors that were modified to respond to the T-REx tetracycline (Tet)-inducible transcription switch. During BAC cloning and mutagenesis in E. coli, not only deletions but also defined mutations of the HSV-1 genome were observed. Successful virus reconstitution from BACs with large inserts demonstrated that HSV-1 has a packaging capacity for foreign sequences of at least 8.1% of its genome size. Targets for Tet-regulated gene expression were the viral DNA polymerase gene (pol) and a reporter gene of glycoprotein B fused to enhanced green fluorescent protein (gBGFP). Results with the pol gene as target showed that virus plaque production could not be significantly controlled by the T-REx gene switch using vectors encoding one copy of the tetR gene. In contrast, an efficient Tet-response was achieved with the gBGFP reporter, which was optimal in a Tet repressor (TetR)-producing cell line, demonstrating that the TetR concentration provided by the virus was not sufficient for a tight control of Tet-regulated gene expression.
Knopf
Charles Werner
CW
Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, DKFZ - ATV, INF 242, 69120 Heidelberg, Germany. c.knopf@dkfz.de
Zavidij
Oksana
O
Rezuchova
Ingeborg
I
Rajcáni
Július
J
D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013754:Tetracyclines / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics
D011993:Recombinant Fusion Proteins; D013754:Tetracyclines; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins