Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis.
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.
D000818:Animals; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D005796:Genes; D005814:Genes, Viral; D007739:L Cells / Q000201:enzymology; D051379:Mice; D010957:Plasmids; D012326:RNA Splicing; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics; D013937:Thymidine Kinase / Q000172:deficiency / Q000235:genetics*; D014158:Transcription, Genetic
D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes