High level gene expression in mammalian cells by a nuclear T7-phage RNA polymerase

Abstract

Here we describe a novel expression system for mammalian cells which is based on transcription of hybrid genes containing T7 phage promoters by a T7 phage RNA polymerase targeted to the nucleus of the host cells. The RNA polymerase gene of T7 phage has been modified by substituting a sequence encoding the nuclear location signal of SV40 large T antigen for the N-terminal part of the polymerase gene. Expression of the modified gene is driven by the mouse metallothionein promoter in transfected mouse Ltk- cells resulting in high concentration of the polymerase in the nucleus. Nuclear T7 RNA polymerase directs efficient transcription of the cat gene under control of a T7 promoter. T7 constructs are expressed at a level at least 6 fold higher than the prototype pRSVcat. The unique properties of this heterologeous expression system are discussed.

Selection of Efficient Cleavage Sites in Target RNAs by Using a Ribozyme Expression Library

“…The expression unit for the ribozyme library should guarantee highlevel expression and stability of the expressed RNA (ribozyme) molecules. For high-level expression, we decided to use alternatively either transcription by pol III or our previously developed T7 promoter-polymerase system for mammalian cells (28)(29)(30). As a gene for embedding the antisense-ribozyme sequences, we chose the human adenovirus type 2-associated vaRNA I gene.…”

“…The corresponding RNAs were called va, vaL, and vaLRz. Plasmid pMTT7N contains the gene for T7 phage RNA polymerase, modified at the 5Ј end with the nuclear localization signal of simian virus 40 T antigen under the control of the mouse metallothionein promoter (28).…”

“…Plasmids were purified by two rounds of cesium chloride gradient centrifugation. For transfection of CHO cells, a modification of the standard calcium phosphate coprecipitation method was used (28). DNA (10 g) in 220 l of H 2 O was mixed with 30 l of 2 M CaCl 2 , and 250 l of 2ϫ HBS (50 mM HEPES, 280 mM NaCl, 1.5 mM sodium phosphate [equal amounts of mono-and dibasic] [pH 6.96]) was added dropwise while the mixture was vortexed.…”

Production of recombinant antibodies in lymphoid and non‐lymphoid cells

“…The recombinant Ig genes may be expressed in lymphoid plasmacytoma cells and in alien systems [ 16,171. Earlier [ 18-201 a high level of the marker gene, chloramphenicol acetyltransferase (CAT), expression was shown in eukaryotic cells under the control of the 'nuclear' T7 RNA polymerase. We were interested in the possibility of expressing the Ig gene tandem in conditions similar to those described in [18]. In the present work a complete tandem of immunoglobulin genes was created consisting of variable (V) genes of light (L) and heavy (H) chains obtained from the mouse hybridoma, and of genes of human constant (C) chains.…”

“…Both cell lines contain in their genome a semisynthetic gene of T7 RNA polymerase [18] and steadily express this enzyme. An earlier modification of the polymerase [18] consisted of replacing the enzyme's N-terminal part with a synthetic oligonucleotide sequence coding for amino acids 124 133 of SW40 viral large T antigen.…”