In our study, stable BHK-21 cell lines expressing targeting shRNAs were selected via FACS sorting for eGFP, which is co-expressed in H1 Lenti-virus vector. Thus, to evaluate efficiency of any given candidate RNAi, the stable clones should be BAY 43-9006 established in order to achieve a high level of anti-virus activity. In this study, we transfected RNAi-VP4 into bovine fetal fibroblasts cells, followed by transfering the transgenic cells into enucleated oocyte cytoplasts, selection of reconstructed embryos were selected based on their expression of eGFP and finally transferring the reconstructed embryos to synchronized recipient cows. Since the major focus of our study is to evaluate the efficiency of FMDV shRNA targeting after transgenic delivery, we used 4-month-old fetuses, insteading of adult animals, for the sake of saving time and money. We confirmed shRNA integration into chromosome of cloned fetuses by Southern Blotting and the expression of shRNA by Northern. Since four-month-old transgenic fetuses could not survive in vitro, for FMDV challenge assay, we used primary tongue epithelium cells established from small pieces of the mucosa collected from the tip of bovine tongue. Since the targeting sequence of RNAi-VP4 was conserved among O, A, and Aisa1 serotypes of FMDV, and ASIA1/YS/CHA/05 strain is able to grow well in BHK-21 cells and in primary tongue epithelium cells, we used ASIA1/YS/CHA/05 strain as challenge virus in this study. We found that shRNA expressed in transgenic fetuses could significantly degrade viral RNA after inoculation of FMDV at a titer of 100 TCID50, and inhibited viral replication. Thus, primary transgenic bovine fetus tongue epithelium cells became much more resistant to FMDV challenge. The most important threat caused by FMDV is the high speed of viral replication, short incubation time, and high contagiousness. Thus, although protective immune responses against FMDV can be efficacious, the rapidity of virus replication and spread can outpace the development of immune defenses and overrun the immune system. Our observation that shRNA inhibited over 91% of viral replication at 48 h after challenge suggest that RNAi-based virus targeting is useful for transgenic cows to get more time to develop immune defense. Needless to say, whether transgenic cows indeed become resistant to FMDV infection will wait for the future study using adult transgenic cows upon FMDV challenge. In fact, we have so far obtained on six-month old male transgenic dairy cattle. Due to their high degree of sequence specificity, shRNAs become ineffective in the presence of escape mutations within and outside the targeted regions, and effective silencing of a single viral gene does not always translate into antiviral effect due to genetic compensation or redundancy. Furthermore, variations within multiple regions of the quasispecies of FMDV were retrospectively revealed by sequencing of FMDV genes, strategies to inhibit RNA virus multiplication based on the use of siRNAs have to consider the high genetic polymorphism exhibited by this group of virus. Thus, it may be important to use multiplex shRNAs if RNAi is to be developed for therapeutic use. In this study, we used shRNAs targeting of viral genes VP2, VP3, and VP4, and observed a significant inhibition of FMDV. Combination of these shRNAs may be necessary to avoid the evolution of escape variants. In conclusion, we obtained three transgenic fetuses expressing RNAi-VP4 against FMDV. Study using primary tongue epithelium cells derived from these fetuses reveal that RNAi-VP4 degraded viral RNA and inhibited viral replication.