Regulated expression of functional external guide sequences in mammalian cells using a U6 RNA polymerase III promoter

• Published in: RNA (Cambridge) 2005-10, Vol.11 (10), p.1588-1595
• Main Authors: Kovrigina, Elizaveta, Yang, Li, Pfund, Emmanuel, Altman, Sidney
• Format: Article
• Language: English
• Subjects: Animals; Blotting, Western; Cell Culture Techniques; Cell Line; Clone Cells; Ecdysterone - analogs & derivatives; Ecdysterone - pharmacology; Electrophoresis, Agar Gel; Gene Expression Regulation, Enzymologic; Humans; Kinetics; Lamin Type A - genetics; Lamin Type A - metabolism; Method; Mice; Promoter Regions, Genetic; Protein Subunits - chemistry; Protein Subunits - genetics; Reverse Transcriptase Polymerase Chain Reaction; Ribonuclease P - antagonists & inhibitors; Ribonuclease P - genetics; Ribonuclease T1 - pharmacology; RNA Polymerase III - chemistry; RNA Polymerase III - genetics; RNA, Messenger - metabolism; Transcription, Genetic - drug effects
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1261/rna.2140505

A regulatable promoter has been stably integrated into a human embryonic kidney cell line. The promoter is a pol III mouse promoter and is under the control of ponasterone A, an ecdysone inducer. The promoter controls transcription of an external guide sequence (EGS) targeted against Rpp38, a protein subunit of human RNase P, or of lamin A/C, a gene product located in the nucleus. The amounts of protein of both gene products are severely reduced when the EGSs are made. Several other, but not all, of the protein subunits of RNase P are also inhibited in both mRNA and protein levels when Rpp38 mRNA is targeted.