216 COMPLETE REDUCTION OF p53 EXPRESSION BY RNA INTERFERENCE FOLLOWING HETEROZYGOUS KNOCKOUT IN PORCINE FIBROBLASTS

Abstract

Because p53 has a critical role in regulation of cell cycle and apoptosis in mammals, mutations of p53 often cause various cancers in mammals. Murine models have contributed to our understanding in cancer related to p53 mutations. Mice, however, have different characteristics from humans in many ways. For instance, the short lifespan of the mouse gives rise to limitations in clinical application of the data obtained from this species. Hence, it would be beneficial to establish a more suitable model in species other than the mouse. The porcine model could be an appropriate alternative because pigs share many anatomical and physiological similarities with humans. However, the production of pigs with homozygous knockout (KO) requires years of breeding heterozygous KO animals. Here, we completely reduced the expression level of p53 mRNA and protein in miniature pig fetal fibroblasts using a combination of gene targeting and RNA interference technique. These cells may be used for nuclear transfer to directly produce pigs without expression of the gene of interest. First, we disrupted the exon 2 region of p53 gene to produce p53 heterozygous KO cells. Miniature pig fetal fibroblasts were transfected with the p53 gene targeting vector. After Geneticin treatment for 2 weeks, a total of 48 surviving colonies were screened by PCR, and one was identified as a homologous recombinant (1/48, 2.08%). Second, the p53 shRNA expression vector was introduced into fibroblasts to isolate p53 knockdown (KD) cells. Transfected fibroblasts were treated with Zeocin for 3 weeks, and the shRNA integrations were confirmed by PCR. We obtained p53 KO, KD, and KOKD fibroblasts which involve p53 KO and KD either separately or simultaneously. In mRNA expression based on RT-PCR, p53 KO fibroblasts showed no difference with wild-type control (91.8 v. 100%). However, the expression levels of KD and KOKD cells significantly decreased (35.5 and 34.7%) compared with the control. In p53 protein levels analysed by Western blot, reduction of the protein was observed in p53 KD, whereas no reduction in p53 KO that might be due to heterozygous mutation. Interestingly, no p53 protein was detected in KOKD, suggesting complete reduction of the protein by synergistic effect of KO and KD. In this study, we demonstrated that various expression levels of p53 in porcine fibroblasts could be achieved by gene targeting and RNA interference technique. Moreover, complete abolishment of protein expression is feasible using combination of the 2 techniques. In further research, cloned miniature pigs with various levels of p53 expression or even lacking p53 expression may be produced using the fibroblasts isolated in the present study as nuclear donors. These pigs may provide new animal models and insights in cancer with respect to the effect of p53.