Altering milk composition: Application of inducible RNAi for controlled gene expression in livestock species
Final Report Abstract
This project examined the use of RNA interference (RNAi) as a means of gene down regulation in livestock. We provide the first comparison of shRNA and miRNA constructs for gene knockdown in livestock. To select the most effective RNAi constructs, each candidate sequence was assessed in several in vitro assays. The results of different assays were however sometimes at variance, indicating that no one assay can be relied upon to predict the effectiveness of an RNAi construct. Our findings are that screening of RNAi constructs is probably best carried out in early primary cells that express the target gene. During the course of the project, porcine p53 was included as an alternative target gene to whey acid protein. We achieved significant down regulation of both porcine p53 and BLG using ShRNA and miRNA constructs. Importantly, the use of miRNA constructs makes tissue specific gene knockdown in large animals a realistic possibility. We show that adipose tissue derived MSCs transgenic for miRNA constructs against BLG can support fetal development Expression analysis of BLG knockdown in the milk of transgenic animals is still outstanding, but nuclear transfer will soon be carried out to produce female animals that will allow later analysis of mammary development, lactation and infant nutrition. In the near future we will also proceed to nuclear transfer using porcine adipose derived mesenchymal stem cells exhibiting p53 knockdown. It will be especially interesting to investigate whether p53 knockdown from a ubiquitous promoter in pigs replicates the human cancer susceptibility condition Li-Fraumeni syndrome, known to be due to loss of p53 function. This project has contributed to the development of RNAi technology in livestock. Results will soon be submitted for publication. This project has contributed to the development of young scientists resulting in several Bachelor's and Master's theses and one Doctoral thesis. The foundation has been laid for continuing work in the manipulation of milk composition for human consumption and the production of large animal models of human cancer. p53 knockdown has recently been shown to play a role in the reprogramming of differentiated cells to more primitive cell types. This project may therefore also facilitate research into new areas such as the efficient derivation of porcine induced pluripotent stem cells.
