High level, regulated expression of the chimeric P-enolpyruvate carboxykinase (GTP)-bacterial O6-alkylguanine-DNA alkyltransferase (ada) gene in transgenic mice.

Abstract

Transgenic animals expressing genes capable of repairing DNA may be a valuable tool to study the effect of DNA-damaging agents on tissue-specific carcinogenesis. For this reason, we constructed a chimeric gene consisting of the promoter-regulatory region of the phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) gene linked to the Escherichia coli ada gene coding for O6-alkylguanine-DNA alkyltransferase and the polyadenylate region from the bovine growth hormone gene. The PEPCK promoter results in gene expression in liver and kidney and is induced by hormones, and its transcription is regulated by diet. The chimeric PEPCK ada gene was injected into the male pronucleus of fertilized eggs to produce transgenic mice. Six of 65 developing mice contained 5-10 copies of the intact trans gene per genome. Two founders transmitted the trans gene in a heterozygous manner, whereas 3 transmitted as germ line mosaics and 1 did not transmit to F1 offspring. All F1 offspring carrying the PEPCK ada trans gene expressed ada mRNA in liver and kidney and produced a functional alkyltransferase with a protein molecular weight of 39,000 originating from the bacterial gene. Total alkyltransferase activity was increased in the liver of F1 offspring from all founder mice, but offspring of only one founder had elevated renal alkyltransferase levels. A diet high in protein markedly increased ada mRNA and alkyltransferase activity within 1 week in both liver and kidney, whereas a high carbohydrate diet for 1 week markedly reduced expression of PEPCK ada and alkyltransferase levels. Nontransgenic animals were unaffected by these dietary manipulations. During induction with a high protein diet, hepatic alkyltransferase in transgenic mice was 16.6 +/- 1.5 units/micrograms DNA (mean +/- SE) compared to 5.3 +/- 0.6 units/micrograms DNA in control animals. This level of alkyltransferase is higher than that in any mammalian tissue noted previously except human liver. Transgenic animals expressing high levels of alkyltransferase should help define the role of DNA repair in protection from carcinogenesis induced by N-nitroso compounds.