Generation of cells expressing improved doxycycline-regulated reverse transcriptional transactivator rtTA2S-M2

Nat Protoc. 2006;1(2):803-11. doi: 10.1038/nprot.2006.117.


Tet-on cell lines engineered to stably express doxycycline (Dox)-regulated reverse transcriptional transactivator (rtTA) have many applications in biomedical research and biotechnology. Unfortunately, construction and maintenance of such cells often proves to be costly, labor intensive and ineffective. Moreover, the Tet-on clones generated using standard methodology were often unstable and frequently displayed significantly changed physiological properties compared with their parental cells. Here we describe an optimized protocol for generation of Tet-on cells. The protocol is based on the use of a recently developed pN1p beta actin-rtTA2S-M2-IRES-EGFP vector (where IRES is an internal ribosome entry site) and permits relatively inexpensive construction of many Tet-on clones with essentially 100% efficiency. The method is well suited for 'difficult' cell lines displaying genetic instability and high levels of epigenetic silencing. The constructed Tet-on cells remain stable with time in the absence of any selection agents, are easy to monitor and preserve the characteristics of parental cells. The protocol can be completed in 2 months.

MeSH terms

  • Cell Line
  • Doxycycline / pharmacology*
  • Electroporation
  • Flow Cytometry
  • Gene Expression Regulation / drug effects*
  • Genetic Engineering / methods*
  • Humans
  • Plasmids
  • Trans-Activators / genetics
  • Trans-Activators / metabolism*
  • Transfection


  • Trans-Activators
  • Doxycycline