Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis

Cell. 1984 Apr;36(4):1007-15. doi: 10.1016/0092-8674(84)90050-3.

Abstract

As an alternative approach to classical genetic analysis, we are investigating the potential of anti-sense (nonsense) DNA strand transcription to inhibit gene activity. A promoter will direct transcription of the complementary nonsense DNA strand when the protein coding sequence of a cloned gene is excised and then reinserted in reverse orientation. When such flipped gene constructions of the HSV thymidine kinase (TK) gene are coinjected with the wild-type gene at a 100:1 ratio, there is a reduction of transient TK expression in TK- mouse L cells. The proportion of viable cells with demonstrable TK activity drops 4-fold as compared with neighboring cells coinjected with TK and an excess of control plasmid. Furthermore, autoradiography of the cells still expressing TK shows that 3H-thymidine incorporation is reduced. Cells contransformed with flipped TK gene constructions have a reduced capacity to express subsequently microinjected TK genes, suggesting that the anti-message phenomenon is due to a trans-inhibition of TK and is probably not an artifact of rearrangements following microinjection.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cloning, Molecular
  • DNA Restriction Enzymes
  • Genes*
  • Genes, Viral*
  • L Cells / enzymology
  • Mice
  • Plasmids
  • RNA Splicing
  • Simplexvirus / enzymology*
  • Simplexvirus / genetics
  • Thymidine Kinase / deficiency
  • Thymidine Kinase / genetics*
  • Transcription, Genetic*

Substances

  • Thymidine Kinase
  • DNA Restriction Enzymes