Suppression of Nonsense Mutations in Cell Culture and Mice by Multimerized Suppressor tRNA Genes

Abstract

We demonstrate here the first experimental suppression of a premature termination codon in vivo by using an ochre suppressor tRNA acting in an intact mouse. Multicopy tRNA expression plasmids were directly injected into skeletal muscle and into the hearts of transgenic mice carrying a reporter gene with an ochre mutation. A strategy for modulation of suppressor efficiency, applicable to diverse systems and based on tandem multimerization of the tRNA gene, is developed. The product of suppression (chloramphenicol acetyltransferase) accumulates linearly with increases in suppressor tRNA concentration to the point where the ochre-suppressing tRNA^Ser is in four- to fivefold excess over the endogenous tRNA^Ser. The subsequent suppressor activity plateau seems to be attributable to accumulation of unmodified tRNAs. These results define many salient variables for suppression in vivo, for example, for tRNA suppression employed as gene therapy for nonsense defects.

ACKNOWLEDGMENTS

This work was supported by the Muscular Dystrophy Association (M.B.) and NIHHL50560 to L.A.L.

We thank Olke Uhlenbeck and Bob Thompson for helpful suggestions and discussion and Tom Cech and Mike Yarus for critical reading of the manuscript. We also thank Uttam RajBhandary for providing the pSV1GT3-ser ochre and pRSVcat(oc27) plasmids and Karen Vikstrom for production of the α-myosin heavy chain–CAT ochre gene-expressing mice.