Arylamine N-acetyltransferase (NAT) genes were targeted for inhibition using short hairpin RNA (shRNA) using two different RNA polymerase III promoters. Constructs were developed for NAT1 and NAT2, the endogenous mouse genes, and for human NAT1. There were fetal and neonatal deaths with these constructs, perhaps due in part to an interferon response as reflected in increases in oligoadenylate synthetase I mRNA levels. Seven out of 8 founders with the U6 promoter generated offspring but only 2 gave positive offspring. Out of 15 founders for H1 promoted constructs, only 4 had positive offspring. When transgenic lines were successfully established, the expression of the targeted genes was variable between animals and was not generally inhibitory.