Lentiviral vectors have become an important and efficient molecular biology tool to integrate foreign DNA into target genomes. These vectors have been previously used in our laboratory to make cloned transgenic fetuses expressing short-hairpin RNAs (shRNAs) targeting the caprine prion mRNA (Golding et al. 2006 Proc. Natl. Acad. Sci. USA 103, 5285-5290) and bovine myostatin mRNA. Specially designed shRNAs have a robust ability to decrease protein expression by initiating a mRNA destruction pathway or by translational inhibition. However, initial experiments targeting foot and mouth (FMDV) viral RNA have indicated that polymerase (Pol) II promoters may be unable to produce enough mature shRNA particles to significantly knock down viral replication in vitro. The goal of this research project was to identify and utilize bovine Pol III promoters to express shRNAs in lentiviral vectors and to express multiple unique shRNAs from a single lentiviral vector using different Pol III promoters. This goal is particularly important to the successful reduction of FMDV replication in a cell, as it limits random mutations from escaping the shRNA-mediated viral genome destruction. The 3 bovine Pol III promoters we selected were 7sk, U6-2, and H1. They were individually amplified from the same genomic DNA preparation. The promoters were inserted immediately upstream of our shRNA expression sequence, resulting in lentiviral vectors designated GT-b7sk, GT-bU6-2, and GT-bH1.To confirm that the promoters were functional, a luciferase reporter assay was performed in HEK 293T cells, where each vector expressed either a shRNA targeting luciferase (luc) or a non-specific shRNA.All promoters expressing luc shRNA resulted in significant reduction of luciferase activity between 68 and 80% compared with non-targeting controls. In addition, there was no significant difference between Pol III promoters when analyzing reduced luciferase activity. In the second phase of the study, we developed 7 unique combinations of 2 or 3 Pol III shRNA expression cassettes to test individual shRNA function with one shRNA designed to target luciferase and the others non-targeting. In multiple Pol III expression constructs, the U6-2 and 7sk promoters resulted in the greatest reduction of luciferase activity at 89 and 95%, respectively. In addition, luciferase activity was reduced to the greatest extent when the luc shRNA was expressed from the second (82%) or third (87%) Pol III cassette. Overall, bovine Pol III-based promoters are effective at expressing shRNAs from a lentiviral vector. In addition, multiple Pol III shRNA expression cassettes can be inserted into a single lentiviral vector and still achieve significant reduction of target protein. These vectors will be used to create transgenic cattle and pigs that express multiple shRNAs targeting the FMDV genome with hopes of creating animals that are resistant to FMDV.